Branched  alpha-glucan, alpha-glucosyltransferase which forms the glucan, their preparation and uses

ABSTRACT

The present invention has objects to provide a glucan useful as water-soluble dietary fiber, its preparation and uses. The present invention solves the above objects by providing a branched α-glucan, which is constructed by glucose molecules and characterized by methylation analysis as follows:
         (1) Ratio of 2,3,6-trimethyl-1,4,5-triacetyl-glucitol to 2,3,4-trimethyl-1,5,6-triacetyl-glucitol is in the range of 1:0.6 to 1:4;   (2) Total content of 2,3,6-trimethyl-1,4,5-triacetyl-glucitol and 2,3,4-trimethyl-1,5,6-triacetyl-glucitolis 60% or higher in the partially methylated glucitol acetates;   (3) Content of 2,4,6-trimethyl-1,3,5-triacetyl-glucitol is 0.5% or higher but less than 10% in the partially methylated glucitol acetates; and   (4) Content of 2,4-dimethyl-1,3,5,6-tetraacetyl-glucitol is 0.5% or higher in the partially methylated glucitol acetates; a novel α-glucosyltransferase which forms the branched α-glucan, processes for producing them, and their uses.

TECHNICAL FIELD

The present invention relates to a branched α-glucan, anα-glucosyltransferase which forms the branched α-glucan, theirpreparation and uses. More particularly, the present invention relatesto a branched α-glucan, which is constructed by glucose molecules andcharacterized by methylation analysis as follows:

(1) Ratio of 2,3,6-trimethyl-1,4,5-triacetyl-glucitol to2,3,4-trimethyl-1,5,6-triacetyl-glucitol is in the range of 1:0.6 to1:4;

(2) Total content of 2,3,6-trimethyl-1,4,5-triacetyl-glucitol and2,3,4-trimethyl-1,5,6-triacetyl-glucitolis 60% or higher in thepartially methylated glucitol acetates;

(3) Content of 2,4,6-trimethyl-1,3,5-triacetyl-glucitol is 0.5% orhigher but less than 10% in the partially methylated glucitol acetates;and

(4) Content of 2,4-dimethyl-1,3,5,6-tetraacetyl-glucitol is 0.5% orhigher in the partially methylated glucitol acetates; anα-glucosyltransferase which forms the above branched α-glucan bytransferring α-glucose residue when allowed to act on maltose and/orα-1,4 glucan having a glucose polymerization degree of 3 or higher;their preparation; a composition comprising the branched α-glucan; andits uses.

BACKGROUND ART

The term, “dietary fiber”, inherently means cell components of plants,which is hardly digestible by animals, such as cellulose, lignin,hemicellulose, pectin, etc., however, in the broad sense, it includeslow-digestible water-soluble polysaccharides which are not digested byamylases. Such water-soluble polysaccharides are called as“water-soluble dietary fiber” (hereinafter, simply abbreviated as“WSDF”, in this specification). Recently, dietary fiber attractsattention to its functions as prebiotics improving bacterial flora inthe intestine in addition to its inherent functions of regulating thefunctions of the intestine, lowering blood-cholesterol level, andcontrolling blood-sugar level. However, it is generally recognized thatdietary fiber and calcium are nutritional elements which areinsufficiently taken in Japanese dietary life. It has been pointed outthat, in the present-day, the average intake of WSDF in Japanese is only50 to 80% of the objective intake, 20 to 25 g/day, recommended in“Nutritional Requirement in Japanese”, 5th edition (1994) (Ref. “MarketTrend of Dietary Fiber”, Shokuhin-To-Kaihatsu (Food processing andingredients), Vol. 34, No. 2, pp. 0.24-27 (1999) (in Japanese)). Underthese circumstances, various low-digestible polysaccharides, which canbe used as materials for various foods and beverages and useful as WSDF,have been proposed.

For example, polysaccharides present in nature or their modifiedproducts, such as low-digestible starch (moist heat treated high-amylosecorn starch), guar gum hydrolyzate, glucomannnan, and low-molecularweight alginate are commercially available as WSDFs. However, since theyhave relatively high viscosities and defects of deteriorating relish andtexture when they are incorporated into foods and beverages, theirusesarerestrictedtoanarrowrange. While, “POLYDEXTROSE®” (developed byPfizer Inc., USA) and low-digestible dextrins are widely utilized in thefield of foods and beverages as WSDF with low-viscosity. “POLYDEXTROSE®”is a synthetic polysaccharide obtained by the steps of heating glucose,sorbitol, and citric acid under a high-vacuum condition; andpolymerizing them by the chemical reaction. It is known that“POLYDEXTROSE®” has complicated branched structures of binding glucosesvia 1,3-, 1,4-, 1,6-, 1,2,6-, and 1,4,6-glucosidic linkages. On theother hand, the low-digestible dextrin is a synthetic polysaccharidewhose digestibility is lowered by inducing 1,2-, 1,3-, 1,2,4-, and1,3,4-glucosidic linkages, not inherently present in starch, formed bytransglucosylation and reverse-reaction during the chemical hydrolysisof starch. The low-digestible dextrin is produced by the steps of addinga small amount of hydrochloric acid to starch, heating the mixture in apowdery form to obtain roasted dextrin, dissolving the resulting roasteddextrin into water, hydrolyzing the roasted dextrin by admixing withα-amylase, purifying the resulting solution with a low viscosity,concentrating the solution, and drying the dextrin with a spray-dryer.As a low-digestible dextrin, another product, produced by the steps ofallowing glucoamylase to act on the above low-digestible dextrin tohydrolyze the digestible part into glucose, removing the resultingglucose, purifying, and drying the dextrin with spray-dryer to furtherlowering digestibility, has been commercialized. However, since thelow-digestible dextrin can not be obtained in a high yield from materialstarch and it causes color-deterioration easily, these characteristicsare problems on the industrial production of the low-digestible dextrin.It is reported that the newly induced glucosidic linkages in“POLYDEXTROSE®” and the low-digestible dextrin include both α- andβ-anomer forms and the reducing end glucose of those are partiallyconverted into 1,6-anhydro-glucose (Ref. “Low-molecular weightwater-soluble dietary fiber”, part of a series of Science of DietaryFibers, pp. 116-131, published by Asakura Shoten (1997)).

Among the glucosidic linkages (hereinafter, “glucosidic linkage” issimply abbreviated as “linkage” in this specification) which are a modeof binding glucose in glucan, α-1,6 linkage is less hydrolysable byamylase than α-1,4 linkage. Therefore, it is expected that glucan richin α-1,6 linkages can be used as WSDF. For example, dextran, producedfrom sucrose as material by the action of dextransucrase (EC 2.4.1.5)from Leuconostoc mesenteroides belonging to lactic acid bacteria, is aglucan in which glucoses are polymerized by mainly α-1,6 linkages, andmay have branches by α-1, 2 and α-1,3 linkages. In the case of usingdextransucrase from Leuconostoc mesenteroides B-512F, the resultingdextran has α-1,6 linkages in the ratio of 90% or higher in the linkagesof the dextran, and is expected to be a low-digestible glucan. However,dextran can not be obtained in a high yield from sucrose, requirescomplicated purifying procedure because of its high viscosity, anddrives up the cost. Therefore, dextran has not been tried to be used asWSDF.

There has been proposed a method for preparing WSDF by allowing amylaseto act on inexpensive starch to hydrolyze α-1,4 linkage for relativelyincreasing the content of α-1,6 linkages. Japanese Patent Kokai No.11,101/2001 disclosed a method for preparing a branched dextrin in whichthe ratio of α-1,6 linkage to α-1,4 linkage is increased to 10 to 20% bythe steps of allowing α-amylase and β-amylase to act on liquefied starchand collecting the residual dextrin. However, the yield of the brancheddextrin from material starch is relatively low and the lowering of thedigestibility can not be expected because the branched dextrin isproduced by a method of increasing the ratio of α-1,6 linkage whilekeeping the inherent branches (α-1,6 linkages) in starch and removingglucose chain in which glucoses are polymerized via α-1,4 linkages.While, dextrin dextranase (EC 2.1.1.2) has been well known as an enzymewhich acts on partial starch hydrolyzate (dextrin) and induces α-1,6linkages in its molecule (Ref. Kazuya Yamamoto et al., Bioscience,Biotechnology, and Biochemistry, Vol. 56, pp. 169-173 (1992)). Dextrindextranase is an enzyme which acts on partial starch hydrolyzate andforms dextran having a structure of polymerizing glucoses via α-1,6linkages by catalyzing mainly α-1,6 glucosyl-transferring reaction.However, there are problems in the well-known dextrin dextranase fromAcetobacter capsulatum belonging to acetic acid bacteria that the ratioof α-1,6 linkage inducible in the molecule is relatively low (Ref.Masayuki Suzuki et al. Journal of Applied Glycoscience, Vol. 48, No. 2,pp. 143-151 (2001)), and the enzyme is unstable. Therefore, the enzymehas not been used practically. Under these circumstances, a novellow-digestible glucan and a process for producing the same have beenstrongly desired for increasing options of WSDF.

DISCLOSURE OF INVENTION

The objects of the present invention are to provide a glucan useful asWSDF, its preparation and uses.

To solve the above objects, the present inventors have extensivelyscreened microorganisms capable of producing an enzyme which forms abranched α-glucan having a relatively large number of branch by using,as substrates, maltose and/or α-1,4 glucan having a glucosepolymerization degree of 3 or higher. (in this specification, “branch”means glucosidic linkage other than α-1,4 linkage in the glucan.) As aresult, the present inventors isolated microorganisms, PP710 and PP349,from soil samples and found that the microorganisms extracellularyproduce a novel α-glucosyltransferase which forms a branched α-glucanhaving α-1,4, α-1,6, α-1,3, α-1,4,6, and α-1,3,6 linkages in itsstructure when allowed to act on maltose and/or α-1,4 glucan having aglucose polymerization degree of 3 or higher. Further, the presentinventors found that the novel enzyme efficiently produces the branchedα-glucan from α-glucan such as partial starch hydrolyzate and thebranched α-glucan which is constructed by glucose molecules andcharacterized by methylation analysis as follows:

(1) Ratio of 2,3,6-trimethyl-1,4,5-triacetyl-glucitol to2,3,4-trimethyl-1,5,6-triacetyl-glucitol is in the range of 1:0.6 to1:4;

(2) Total content of 2,3,6-trimethyl-1,4,5-triacetyl-glucitol and2,3,4-trimethyl-1,5,6-triacetyl-glucitolis 60% or higher in thepartially methylated glucitol acetates;

(3) Content of 2,4,6-trimethyl-1,3,5-triacetyl-glucitol is 0.5% orhigher but less than 10% in the partially methylated glucitol acetates;and

(4) Content of 2,4-dimethyl-1,3,5,6-tetraacetyl-glucitol is 0.5% orhigher in the partially methylated glucitol acetates.

Also, the present inventors found that a starch-degrading amylase ispresent in the crude preparation of the α-glucosyltransferase, obtainedby culturing PP710, as a concomitant enzyme. It was found that abranched α-glucan with a relatively higher WSDF content can be producedby using the crude enzyme preparation, or the purified amylase andα-glucosyltransferase in combination, in comparison with the case ofusing α-glucosyltransferase only. Further, it was found that theweight-average molecular weight and the WSDF content of the branchedα-glucan can be controlled by using the α-glucosyltransferase togetherwith well-known amylases and starch-debranching enzymes as a substituentof the amylase. In addition, the present inventors found that thebranched α-glucan, obtainable by the above methods, shows a relativelyhigher ratio of α-1,6 linkage than the material α-1,4 glucan; asignificantly low-digestibility which is useful as WSDF; and effects ofinhibiting the elevation of blood-sugar level and lowering lipids inliving bodies. Based on the above knowledge, the present inventorsaccomplished the present invention.

The present invention solves the above objects by providing a branchedα-glucan which is constructed by glucose molecules and characterized bymethylation analysis as follows:

(1) Ratio of 2,3,6-trimethyl-1,4,5-triacetyl-glucitol to2,3,4-trimethyl-1,5,6-triacetyl-glucitol is in the range of 1:0.6 to1:4;

(2) Total content of 2,3,6-trimethyl-1,4,5-triacetyl-glucitol and2,3,4-trimethyl-1,5,6-triacetyl-glucitol is 60% or higher in thepartially methylated glucitol acetates;

(3) Content of 2,4,6-trimethyl-1,3,5-triacetyl-glucitol is 0.5% orhigher but less than 10% in the partially methylated glucitol acetates;and

(4) Content of 2,4-dimethyl-1,3,5,6-tetraacetyl-glucitol is 0.5% orhigher in the partially methylated glucitol acetates; a novelα-glucosyltransferase which forms the branched α-glucan; theirpreparation and uses.

According to the present invention, a branched α-glucan, having whitecolor, low digestibility, and usefulness as WSDF, can be produced in ahigh yield, large amount, and low cost, and provided to various fieldsincluding foods and beverages.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a comparison of gel-filtration HPLC chromatograms ofGlucans A and B, respectively prepared from partial starch hydrolyzateby using α-glucosyltransferase from Bacillus circulans PP710 and fromArthrobacter globiformis PP349, and that of partial starch hydrolyzateused as substrate for the enzymes.

FIG. 2 shows the reference diagrams of the structures of partial starchhydrolyzate and the branched α-glucan of the present invention.

FIG. 3 shows the optimum temperature of α-glucosyltransferase fromBacillus circulans PP710.

FIG. 4 shows the optimum pH of α-glucosyltransferase from Bacilluscirculans PP710.

FIG. 5 shows the thermal stability of α-glucosyltransferase fromBacillus circulans PP710.

FIG. 6 shows the pH stability of α-glucosyltransferase from Bacilluscirculans PP710.

FIG. 7 shows the optimum temperature of α-glucosyltransferase fromArthrobacter globiformis PP349.

FIG. 8 shows the optimum pH of α-glucosyltransferase from Arthrobacterglobiformis PP349.

FIG. 9 shows the thermal stability of α-glucosyltransferase fromArthrobacter globiformis PP349.

FIG. 10 shows the pH stability of α-glucosyltransferase fromArthrobacter globiformis PP349.

FIG. 11 shows the optimum temperature of amylase from Bacillus circulansPP710.

FIG. 12 shows the optimum pH of amylase from Bacillus circulans PP710.

FIG. 13 shows the thermal stability of amylase from Bacillus circulansPP710.

FIG. 14 shows the pH stability of amylase from Bacillus circulans PP710.

FIG. 15 shows the comparison of gel-filtration HPLC chromatograms of thebranched α-glucan, prepared by using purified preparations ofα-glucosyltransferase and amylase from Bacillus circulans PP710 incombination, and that of partial starch hydrolyzate used as substratefor the enzymes.

FIG. 16 shows the comparison of gel-filtration HPLC chromatograms of thebranched α-glucan, prepared by using purified preparations ofα-glucosyltransferase from Bacillus circulans PP710 and isoamylase incombination, and that of partial starch hydrolyzate used as substratefor the enzymes.

FIG. 17 shows the comparison of gel-filtration HPLC chromatograms of thebranched α-glucan, prepared by using purified preparations ofα-glucosyltransferase from Bacillus circulans PP710 and α-amylase incombination, and that of partial starch hydrolyzate used as substratefor the enzymes.

FIG. 18 shows the comparison of gel-filtration HPLC chromatograms of thebranched α-glucan, prepared by using purified preparations ofα-glucosyltransferase and from Bacillus circulans PP710 and CGTase incombination, and that of partial starch hydrolyzate used as substratefor the enzymes.

FIG. 19 shows the comparison of gel-filtration HPLC chromatograms of thebranched α-glucan, prepared by using purified preparations ofα-glucosyltransferase from Bacillus circulans PP710, isoamylase, andCGTase in combination, and that of partial starch hydrolyzate used assubstrate for the enzymes.

EXPLANATION OF SYMBOLS In FIG. 1 and FIGS. 15 to 19,

-   -   A: Eluting position corresponding to the molecular weight of        1000,000 daltons    -   B: Eluting position corresponding to the molecular weight of        100,000 daltons    -   C: Eluting position corresponding to the molecular weight of        10,000 daltons    -   D: Eluting position corresponding to the molecular weight of        1,000 daltons    -   E: Eluting position corresponding to the molecular weight of 100        daltons

In FIG. 1,

-   -   a: Gel-filtration HPLC chromatogram of partial starch        hydrolyzate used as substrate    -   b: Gel-filtration HPLC chromatogram of Glucan A    -   c: Gel-filtration HPLC chromatogram of Glucan B    -   1: Position corresponding to the glucose polymerization degree        of 499    -   2: Position corresponding to the glucose polymerization degree        of 6.3    -   3: Position corresponding to the glucose polymerization degree        of 384    -   4: Position corresponding to the glucose polymerization degree        of 22.2    -   5: Position corresponding to the glucose polymerization degree        of 10.9    -   6: Position corresponding to the glucose polymerization degree        of 1    -   7: Position corresponding to the glucose polymerization degree        of 433    -   8: Position corresponding to the glucose polymerization degree        of 22.8    -   9: Position corresponding to the glucose polymerization degree        of 10.9    -   10: Position corresponding to the glucose polymerization degree        of 1

In FIG. 2,

-   -   1: Reference diagram of partial starch hydrolyzate    -   2: Reference diagram of the branched α-glucan of the present        invention    -   a: Non-reducing end glucose residue    -   b: Glucose residue involving α-1,3 linkage    -   c: Glucose residue involving α-1,4 linkage    -   d: Glucose residue involving α-1,6 linkage    -   e: Glucose residue involving α-1,3,6 linkage    -   f: Glucose residue involving α-1,4,6 linkage    -   Diagonal broken line: α-1,3 linkage    -   Horizontal solid line: α-1,4 linkage    -   Vertical solid line: α-1,6 linkage

In FIG. 13,

-   -   •: In the absence of Ca²⁺ ion    -   ∘: In the presence of 1 mM Ca²⁺ ion

In FIG. 15,

-   -   a: Gel-filtration HPLC chromatogram of partial starch        hydrolyzate used as substrate    -   b: Gel-filtration HPLC chromatogram of the branched α-glucan        prepared by using 10 units/g-substrate of α-glucosyltransferase        and 0.1 unit/g-substrate of amylase    -   c: Gel-filtration HPLC chromatogram of the branched α-glucan        prepared by using 10 units/g-substrate of α-glucosyltransferase        and 0.2 unit/g-substrate of amylase    -   d: Gel-filtration HPLC chromatogram of the branched α-glucan        prepared by using 10 units/g-substrate of α-glucosyltransferase        and 0.5 unit/g-substrate of amylase    -   e: Gel-filtration HPLC chromatogram of the branched α-glucan        prepared by using 10 units/g-substrate of α-glucosyltransferase        and 1 unit/g-substrate of amylase

In FIG. 16,

-   -   a: Gel-filtration HPLC chromatogram of partial starch        hydrolyzate used as substrate    -   b: Gel-filtration HPLC chromatogram of the branched α-glucan        prepared by using 10 units/g-substrate of α-glucosyltransferase        and 50 units/g-substrate of isoamylase    -   c: Gel-filtration HPLC chromatogram of the branched α-glucan        prepared by using 10 units/g-substrate of α-glucosyltransferase        and 200 units/g-substrate of isoamylase    -   d: Gel-filtration HPLC chromatogram of the branched α-glucan        prepared by using 10 units/g-substrate of α-glucosyltransferase        and 500 units/g-substrate of isoamylase    -   e: Gel-filtration HPLC chromatogram of the branched α-glucan        prepared by using 10 units/g-substrate of α-glucosyltransferase        and 1,000 units/g-substrate of isoamylase

In FIG. 17,

-   -   a: Gel-filtration HPLC chromatogram of partial starch        hydrolyzate used as substrate    -   b: Gel-filtration HPLC chromatogram of the branched α-glucan        prepared by using 10 units/g-substrate of α-glucosyltransferase        and 0.1 unit/g-substrate of α-amylase    -   c: Gel-filtration HPLC chromatogram of the branched α-glucan        prepared by using 10 units/g-substrate of α-glucosyltransferase        and 0.2 unit/g-substrate of α-amylase    -   d: Gel-filtration HPLC chromatogram of the branched α-glucan        prepared by using 10 units/g-substrate of α-glucosyltransferase        and 0.5 unit/g-substrate of α-amylase    -   e: Gel-filtration HPLC chromatogram of the branched α-glucan        prepared by using 10 units/g-substrate of α-glucosyltransferase        and 1.0 unit/g-substrate of α-amylase

In FIG. 18,

-   -   a: Gel-filtration HPLC chromatogram of partial starch        hydrolyzate used as substrate    -   b: Gel-filtration HPLC chromatogram of the branched α-glucan        prepared by using 10 units/g-substrate of α-glucosyltransferase        and 0.1 unit/g-substrate of CGTase    -   c: Gel-filtration HPLC chromatogram of the branched α-glucan        prepared by using 10 units/g-substrate of α-glucosyltransferase        and 0.2 unit/g-substrate of CGTase    -   d: Gel-filtration HPLC chromatogram of the branched α-glucan        prepared by using 10 units/g-substrate of α-glucosyltransferase        and 0.5 unit/g-substrate of CGTase    -   e: Gel-filtration HPLC chromatogram of the branched α-glucan        prepared by using 10 units/g-substrate of α-glucosyltransferase        and 1.0 unit/g-substrate of CGTase

In FIG. 19,

-   -   a: Gel-filtration HPLC chromatogram of partial starch        hydrolyzate used as substrate    -   b: Gel-filtration HPLC chromatogram of the branched α-glucan        prepared by using 10 units/g-substrate of α-glucosyltransferase,        50 units/g-substrate of isoamylase, and 1 unit/g-substrate of        CGTase    -   c: Gel-filtration HPLC chromatogram of the branched α-glucan        prepared by using 10 units/g-substrate of α-glucosyltransferase,        200 units/g-substrate of isoamylase, and 1 unit/g-substrate of        CGTase    -   d: Gel-filtration HPLC chromatogram of the branched α-glucan        prepared by using 10 units/g-substrate of α-glucosyltransferase,        500 units/g-substrate of isoamylase, and 1 unit/g-substrate of        CGTase    -   e: Gel-filtration HPLC chromatogram of the branched α-glucan        prepared by using 10 units/g-substrate of α-glucosyltransferase,        1,000 units/g-substrate of isoamylase, and 1 unit/g-substrate of        CGTase

BEST MODE FOR CARRYING OUT THE INVENTION

The term, “glucan”, as referred to as in the present invention means anoligosaccharide or polysaccharide, with a glucose polymerization degreeof 3 or higher, which is constructed by glucose molecules. The branchedα-glucan of the present invention is an α-glucan constructed by glucosemolecules and shows the following characteristics by the methylationanalysis:

(1) Ratio of 2,3,6-trimethyl-1,4,5-triacetyl-glucitol to2,3,4-trimethyl-1,5,6-triacetyl-glucitol is in the range of 1:0.6 to1:4;

(2) Total content of 2,3,6-trimethyl-1,4,5-triacetyl-glucitol and2,3,4-trimethyl-1,5,6-triacetyl-glucitolis 60% or higher in thepartially methylated glucitol acetates;

(3) Content of 2,4,6-trimethyl-1,3,5-triacetyl-glucitol is 0.5% orhigher but less than 10% in the partially methylated glucitol acetates;and

(4) Content of 2,4-dimethyl-1,3,5,6-tetraacetyl-glucitol is 0.5% orhigher in the partially methylated glucitol acetates.

The term, “methylation analysis”, as referred to as in the presentinvention means a generally well-known method for determining thelinkages of monosaccharide as the component in poly- oroligo-saccharides. Analysis of glucosidic linkages in a glucan by themethylation analysis is carried out by the following steps of:

-   -   methylating all the free hydroxyl groups of glucose residues        which constitute the glucan;    -   hydrolyzing the completely methylated glucan;    -   reducing the resulting methylated glucoses for eliminating the        anomers to make into methylated glucitols;    -   acetylating the free hydroxyl groups of the methylated glucitols        to make into the partially methylated glucitol acetates        (hereinafter, “partially methylated glucitol acetate” may be        abbreviated as “partially methylated product” by abbreviating        acetylated positions and “glucitol acetate” in this        specification); and analyzing the resulting partially methylated        products by a gas chromatography.

Various partially methylated products, derived from glucose residuesrespectively different in the glucosidic linkage in the glucan, can berepresented by the percentage (%) of peak area per the total peak areaof all the partially methylated products in the gas chromatogram. Then,from the peak area (%), the ratio of the glucose residue different inthe linkage, i.e., the ratio of each glucosidic linkage can bedetermined. In this specification, “ratio” of the partially methylatedproducts is defined as the ratio of peak area in the gas chromatogramobtained by the methylation analysis. Also, “%” of the partiallymethylated products is defined as “peak area %” in the gas chromatogramobtained by the methylation analysis.

2,3,6-Trimethyl-1,4,5-triacetyl-glucitol (hereinafter, abbreviated as“2,3,6-trimethylated product”), in the above (1), means the glucoseresidues whose C-4 position is involved in 1,4 linkage, and2,3,4-trimethyl-1,5,6-triacetyl-glucitol (hereinafter, abbreviated as“2,3,4-trimethylated product”) means the glucose residues whose C-6position is involved in 1,6 linkage. Also, “ratio of 2,3,6-trimethylatedproduct to 2,3,4-trimethylated product is in the range of 1:0.6 to 1:4”means that, in the gas chromatogram of the partially methylated glucitolactates in the methylation analysis, the ratio of glucose residues whoseC-6 and C-1 positions involve the linkage to the total content ofglucose residues whose C-4 and C-1 positions involve the linkage andglucose residues whose C-6 and C-1 positions involve the linkage is inthe range of 37.5 to 80.0%.

“Total content of 2,3,6-trimethylated product and 2,3,4-trimethylatedproduct is 60% or higher in the partially methylated products”, in theabove (2), means that, in the branched α-glucan of the presentinvention, the total content of glucose residues whose C-4 and C-1positions involve the linkage and glucose residues whose C-6 and C-1positions involve the linkage is 60% or higher in all glucose residuesconstituting the glucan.

In the same manner, “2,4,6-trimethyl-1,3,5-triacetyl-glucitol”(hereinafter, abbreviated as “2,4,6-trimethylated product”), in theabove (3), means glucose residues whose C-3 position involves 1,3linkage. Also, “content of 2,4,6-trimethylated product is 0.5% or higherbut less than 10% in the partially methylated products” means that, inthe branched α-glucan of the present invention, the content of glucoseresidues whose C-3 and C-1 positions involve the linkage is 0.5% orhigher but less than 10% in all glucose residues constituting theglucan.

Similarly, “2,4-dimethyl-1,3,5,6-tetraacetyl-glucitol” (hereinafter,abbreviated as “2,4-demethylated product”), in the above (4), meansglucose residues whose C-3 and C-6 positions respectively involve 1,3and 1,6 linkages. Also, “content of 2,4-demethylated product is 0.5% orhigher in the partially methylated products” means that, in the branchedα-glucan of the present invention, the content of glucose residues whoseC-3, C-6, and C-1 positions involve the linkage is 0.5% or higher in allglucose residues constituting the glucan.

The branched α-glucan of the present invention, which fulfills the abovecharacteristics (1) to (4), is a novel glucan hitherto unknown. Theorder of linking glucose residues in the branched α-glucan of thepresent invention is not specifically restricted as far as it fulfillsthe above characteristics (1) to (4) by methylation analysis.

Usually, the branched α-glucan of the present invention is in the formof a mixture of branched α-glucans having various glucose polymerizationdegrees of 10 or higher. The value of dividing the weight-averagemolecular weight (Mw) with the number average molecular weight (Mn),Mw/Mn, of the branched α-glucan of the present invention is, usually,less than 20.

The branched α-glucan of the present invention is characterized in thatisomaltose is formed, usually, in an amount of 25% (w/w) or higher butless than 50% (w/w), on a dry solid basis of the hydrolyzate, whenisomaltodextranase (EC 3.2.1.94), which is an enzyme capable ofhydrolyzing α-1,2, α-1,3, α-1,4, and α-1,6 linkages as far as thelinkage is adjuscent to reducing end side of isomaltose structure in aglucan, is allowed to act on the branched α-glucan of the presentinvention.

The branched α-glucan of the present invention is characterized in thatthe WSDF content is, usually, 40% (w/w) or higher when WSDF isquantified according to the method described in Section 8, Dietaryfiber, (2) High-performance liquid chromatography (Enzyme-HPLC method),“Methods for analyzing nutritional components (Appendix 1-3 of NutritionLabeling Standard) in Nutrition Labeling Standard (Notification No. 146of Ministry of Health, Labour, and Welfare, May, 1986)”. The outline ofthe above high-performance liquid chromatography method (hereinafter,abbreviated as “Enzyme-HPLC method”) is as follows: A sample ishydrolyzed by a series of enzyme-treatments using a thermostableα-amylase, protease, and amyloglucosidase (glucoamylase). Then,proteins, organic acids, and inorganic salts are removed from theresulting enzyme-treated mixture using ion-exchange resins to make intoa sample solution for high-performance liquid chromatography (HPLC).Successively, the sample solution is subjected to gel-filtration HPLCfor measuring peak areas of undigested glucan and glucose in the HPLCchromatogram. Then, the WSDF content of the sample is calculated basedon the peak areas and the amount of glucose in the sample solution,separately determined by conventional glucose oxidase-peroxidase method.The Enzyme-HPLC method is also explained in detail in Experimentsdescribed later.

As shown in Experiment 9 described later, the branched α-glucan of thepresent invention is hardly digested by salivary α-amylase, pancreasα-amylase, and small intestinal α-glucosidase when orally ingested.Accordingly, the branched α-glucan of the present invention can be usedas a low-calorie WSDF with a low-digestibility, which does not stimulatethe rapid elevation of blood-sugar level and the secretion of insulin.In addition, the branched α-glucan has characteristics of not inducingacid fermentation by microorganisms in the mouth and inhibiting theformation of insoluble glucans which is a cause of dental plaque whenused together with sucrose. Therefore, the branched α-glucan of thepresent invention can be advantageously used as a low- oranti-cariogenic saccharide. Further, the branched α-glucan of thepresent invention shows no toxity in the acute-toxity test using mice.

As shown in Experiments 20 and 21 described later, since the branchedα-glucan of the present invention inhibits the elevation of blood-sugarlevel and insulin level when it is ingested together with an amylaseoussubstance, in comparison with the case of ingesting an amylaceoussubstance only, it can be used as an agent for inhibiting the elevationof blood-sugar level.

As shown in Experiment 22 described later, since the branched α-glucanof the present invention inhibits the excess accumulation of lipids inliving bodies, it can be used as an agent for lowering lipids in livingbodies.

In the cases of using the branched α-glucan as the above agent forinhibiting the elevation of blood-sugar level or that for loweringlipids in living bodies, the branched α-glucan with a relatively highWSDF content is preferable to exercise the effects. Therefore, the WSDFcontent of the branched α-glucan is preferable to be, usually, 40% (w/w)or higher, desirably, 50% (w/w) or higher, more desirably, 60% (w/w) orhigher.

“α-Glucosyltransferase” as referred to as in the present invention meansany enzyme which acts on maltose and/or α-1,4 glucan having a glucosepolymerization degree of 3 or higher as substrate and forming thebranched α-glucan of the present invention by catalyzing theglucosyl-transfer without substantial hydrolytic action. Theα-glucosyltransferase of the present invention is different fromwell-known α-glucosidase from fungi and dextrin-dextranase from thegenus Acetobacter in the characteristics of showing weak hydrolyticactivity and efficient transferring activity under low to high substrateconcentration without depending on the substrate concentration, and offorming α-1,3 and α-1,3,6 linkages.

The enzyme activity of the α-glucosyltransferase of the presentinvention can be assayed as follows: A substrate solution is prepared bydissolving maltose in 20 mM acetate buffer (pH 6.0) to give a finalconcentration of 1% (w/v). 0.5 ml of an enzyme solution is added to 5 mlof the substrate solution, and the mixture solution is incubated at 40°C. for 30 min. After the reaction, 0.5 ml of the reaction mixture isadmixed with 5 ml of 20 mM phosphate buffer (pH 7.0) and boiled for 10min to stop the reaction. Successively, the amount of glucose in thesolution is measured by the glucose oxidase-peroxidase method accordingto the conventional method, and the amount of glucose formed in thereaction mixture is calculated. One unit of the α-glucosyltransferaseactivity is defined as the amount of enzyme which forms one μmole ofglucose per minute under the above conditions.

As a concrete example of the α-glucosyltransferase of the presentinvention, the enzyme having the following physicochemical propertiescan be listed.

(1) Molecular weight

-   -   90,000±10,000 daltons when determined on SDS-polyacrylamide gel        electrophoresis;

(2) Optimum temperature

-   -   50 to 55° C. when reacted at pH 6.0 for 30 min;

(3) Optimum pH

-   -   pH 5.0 to 6.3 when reacted at 40° C. for 30 min;

(4) Thermal stability

-   -   Stable up to 40° C. when incubated at pH 6.0 for 60 min; and

(5) pH Stability

-   -   Stable in the pH range of 3.5 to 8.4 when incubated at 4° C. for        24 hours;

As another concrete example of the α-glucosyltransferase of the presentinvention, the enzyme having the following physicochemical propertiescan be listed.

(1) Molecular weight

-   -   90,000±10,000 daltons when determined on SDS-polyacrylamide gel        electrophoresis;

(2) Optimum temperature

-   -   About 50° C. when reacted at pH 6.0 for 30 min;

(3) Optimum pH

-   -   About pH 6.0 when reacted at 40° C. for 30 min;

(4) Thermal stability

-   -   Stable up to 40° C. when incubated at pH 6.0 for 60 min; and

(5) pH Stability

-   -   Stable in the pH range of 4.0 to 8.0 when incubated at 4° C. for        24 hours;

Although the α-glucosyltransferase of the present invention is notrestricted by its source, microorganisms are preferable as the source.Particularly, microorganisms, PP710 and PP349, isolated from soil by thepresent inventors can be preferably used as the source. The followingTables 1 and 2 are the identification results of the strains PP710 andPP349, capable of producing the α-glucosyltransferase. Theidentification of the strains was carried out according to the method asdescribed in “BISEIBUTSU-NO-BUNRUI-TO-DOTEI” (Classification andIdentification of Microorganisms), edited by Takeji Hasegawa, publishedby Japan Scientific Societies Press, Tokyo, Japan (1985).

TABLE 1 <A: Morphology> Characteristic of cells Existing usually in arod shape when incubated at 27° C. of 0.5 × 1.0 to 2.0 × 6.0 μm, innutrient agar Possessing no motility, Forming spores, Gram stain;positive, <B: Cultural property> Characteristics of colony formed whenincubated at 27° C. in nutrient agar plate Shape Circular colony havinga diameter of 1 to 2 mm after 2 days incubation Rim Entire ProjectionSemi-lenticular Gloss Dull Surface Smooth Color Translucence, GrayCharacteristics of colony formed when incubated at 27° C. in nutrientagar slant Growth Medium Shape Thread-like Characteristics of colony Notliquefied formed when incubated at 27° C. in nutrient gelatin stabculture <C: Physiological properties> VP-test Negative Indole formationNegative Dihydroxylacetone formation Negative Hydrolysis of starchPositive Pigment formation Not forming soluble pigments Urease NegativeOxidase Negative Catalase Positive Growing range pH: 5.5 to 10.0,temperature: 15 to 37° C. Formation of acids from D-glucose PositiveFormation of gases from D-glucose Negative Utilization of citric acidPositive Decomposition of tyrosine Negative Deamination of phenylalanineNegative Reduction of nitrate Positive Oxygen requirements AerobicGrowth in the presence of Positive lysozyme Mol % of guanine (G) plus53.4% cytosine (C) of DNA

TABLE 2 <A: Morphology> Characteristic of cells Existing usually in acoccus or when incubated at 27° C. rod shape of 0.4 × 1.0 to in nutrientagar 0.6 × 3.0 μm, Exhibiting polymorphism showing rod-coccus cycle(early phase: rod-shape, late phase: short rod- or coccus-shape),Possessing no motility, Forming spores, Gram stain; positive, <B:Cultural property> Characteristics of colony formed when incubated at27° C. in nutrient agar plate Shape Circular colony having a diameter of1 to 2 mm after 2 days incubation Rim Entire Projection Semi-lenticularGloss Moist gloss Surface Smooth Color Translucence, MaizeCharacteristics of colony formed when incubated at 27° C. in nutrientagar slant Growth Medium Shape Homogenous Characteristics of colony Notliquefied formed when incubated at 27° C. in nutrient gelatin stabculture <C: Physiological properties> Oxygen requirements Aerobic Majordiamino acid in cell wall Lysine Peptideglycan Lysine, Alanine N-Acyltype of cell wall Acetyl Major sugar components D-Galactose, D-Glucoseconstructing cell wall Catalase Positive Extracellular DNase PositiveHydrolysis of starch Positive Vitamin requirement Negative Homology of16S rRNA with that of 97% Arthrobacter globiformis type culture(DSM20124)

The above bacteriological properties of strains PP710 and PP349 werecompared with those of known microorganisms with reference to “Bergey'sManual of Systematic Bacteriology, Vol. 2 (1986)” and “RibosomalDatabase (URL:http://rdp.cme.msu.edu/index.jsp). As a result, it wasrevealed that the strains PP710 and PP349 were respectively identifiedas Bacillus circulans and Arthrobacter globifomis. Based on theseresults, the present inventors named the two strains to “Bacilluscirculans PP710” and “Arthrobacter globiformis PP349”, and depositedthem on Feb. 1, 2006, in International Patent Organism, NationalInstitute of Advanced Industrial Science and Technology, AIST TsukubaCentral 6, 1-1, Higashi 1-Chome Tsukuba-shi, Ibaraki-ken Japan, andaccepted under the accession numbers of FERM BP-10771 and FERM BP-10770,respectively. The microorganisms capable of producing theα-glucosyltransferase of the present invention include the above strainsand their mutants capable of producing the enzyme in large amount, whichare obtainable by inducing mutation to the above strains and screeningthe enzyme-hyper-producing mutants.

Any nutrient culture medium can be used for cultivating anymicroorganism capable of producing the α-glucosyltransferase of thepresent invention as long as it can grow therein and produce theα-glucosyltransferase: For example, synthetic- and natural-culture mediacan be used as nutrient culture media. Any carbon source can be used aslong as it is utilized by the microorganisms: Examples of such carbonsource are saccharides such as starch and phytoglycogen, obtainable fromplants; glycogen and pullulan, obtainable from animals andmicroorganisms; hydrolyzates thereof, glucose, fructose, lactose,sucrose, mannitol, sorbitol, and saccharide syrups; and organic acidssuch as citric acid and succinic acid. The concentrations of thesecarbon sources in nutrient culture media are appropriately chosen. Thenitrogen sources usable in the present invention are, for example,inorganic nitrogen compounds such as ammonium salts and nitrates;organic nitrogen compounds such as urea, corn steep liquor, casein,peptone, yeast extract and beef extract. The inorganic ingredientsusable in the invention are, for example, calcium salts, magnesiumsalts, potassium salts, sodium salts, phosphates, manganese salts, zincsalts, iron salts, copper salts, molybdenium salts, and cobalt salts. Ifnecessary, amino acids and vitamins can be suitably used.

The microorganisms capable of producing the α-glucosyltransferase of thepresent invention are cultured under aerobic conditions, usually, at atemperature in the range of 15 to 37° C. and at a pH in the range of 5.5to 10, preferably, at a temperature in the range of 20 to 34° C. and ata pH in the range of 5.5 to 8.5. The cultivation time is set to a timelonger than that required for the growth of the microorganisms,preferably, 10 to 150 hours. The concentration of dissolved oxygen isnot specifically restricted, but usually, 0.5 to 20 ppm. Theconcentration of dissolved oxygen can be kept within the above range bycontrolling aeration and agitation. The cultivation can be carried outbatch-wise or in a continuous manner.

After culturing the microorganisms capable of producing theα-glucosyltransferase according to the method described above, theculture containing the enzyme of the present invention is recovered. Themajor activity of the α-glucosyltransferase is found in the cell-freesupernatant in both cases of Bacillus circulans PP710, FERM BP-10771,and Arthrobacter globiformis PP349, FERM BP-10770. Both the cell-freesupernatant and the culture broth can be used as a crude enzymepreparation. Conventional liquid-solid separation methods can beemployed to remove cells from the culture. For example, methods todirectly centrifuge the resultant culture, as well as those to filtratethe culture with pre-coated filters or to separate cells by membranefiltration using plane filters or follow fibers, can be suitably used.While cell-free supernatants thus obtained can be used intact as a crudeenzyme solution, they can be concentrated prior to use. Theconcentration methods usable in the invention are, for example, saltingout using ammonium sulfate, sedimentation using acetone or alcohol, andconcentration using membranes such as plane filters and follow fibers.

The α-glucosyltransferase can be subjected to the conventionalimmobilization using cell-free supernatants and their concentrates.Examples of such conventional methods are conjugation methods using ionexchangers, covalent bindings and adsorptions using resins andmembranes, and inclusion methods using high molecular weight substances.

As described above, a crude enzyme solution can be used intact afterconcentrating it as the α-glucosyltransferase of the present invention.If necessary, the enzyme can be advantageously used after separating orpurifying the crude enzyme solution by suitable conventional methodsused in the art, for example, salting out, ion-exchange chromatography,hydrophobic chromatography, gel-filtration chromatography, affinitychromatography, preparative electrophoresis, etc.

α-1,4 Glucan having a glucose polymerization degree of 3 or higher,which can be used as a substrate for the α-glucosyltransferase of thepresent invention, includes starch, amylose, amylopectin, glycogen, andtheir partial hydrolyzates such as amylodextrins, maltodextrins,maltooligosaccharides, obtainable by partially hydrolyzing them withamylases and acids. The partial hydrolyzates obtainable by hydrolyzingstarch, amylose, amylopectin, and glycogen by using amylase such asα-amylase (EC 3.2.1.1), β-amylase (EC 3.2.1.2), maltotetraose-formingamylase (EC 3.2.1.60), maltopentaose-forming amylase,maltohexaose-forming amylase (EC 3.2.1.98), cyclomaltodextringlucanotransferase (EC 2.4.1.19, hereinafter abbreviated as “CGTase” inthis specification), etc., described in “Handbook of Amylases andRelated Enzymes” published by Pergamon Press Inc., (Tokyo), 1988; can beused as the partial hydrolyzates. Further, starch-debranching enzymessuch as pullulanase (EC 3.2.1 41) and isoamylase (EC 3.2.1.68) can bearbitrarily used for preparing the partial hydrolyzates. Bothsubcelestal starches such as those from corn, wheat, rice, etc., andsubterranean starches such as those from potato, sweet potato, tapioca,etc., can be used as amylaceous substrates. The substrate can bepreferably used in the form of a solution prepared by gelatinizingand/or liquefying the above starch. Further, chemically modified starch,obtained by chemically modifying a part of starch, such as etherifiedstarch (hydroxypropyl-starch, carboxymethyl-starch, acetyl-starch,etc.), esterified starch (phosphorylated starch, octernylsuccinate esterof starch, etc.), cross-linked starch (starch cross-linked byacetyladipate, starch cross-linked by phosphate, starch cross-linked byhydroxypropylphosphate, etc.), etc, can be used as a substrate of theα-glucosyltransferase of the present invention.

When the α-glucosyltransferase of the present invention is allowed toact on a substrate, the substrate concentration is not specificallyrestricted. For example, the reaction by the α-glucosyltransferase ofthe present invention proceeds to form the branched α-glucan even in thecase of using a substrate solution with a relatively low concentrationsuch as 0.5% (w/v). For industrial production, the substrateconcentration is preferable to be, usually, 1% (w/v) or higher,preferably, 5 to 60% (w/v), more preferably, 10 to 50% (w/v); and thebranched α-glucan of the present invention can be advantageouslyproduced under the condition. The reaction temperature used in thepresent enzymatic reaction can be set to a temperature at which thereaction proceeds, i.e., a temperature up to about 60° C., preferably, atemperature in the range of 30 to 50° C. The reaction pH is controlledin the range of, usually, 4 to 8, preferably, 5 to 7. Since the amountof enzyme and the reaction time are closely related, the conditions areadequately chosen with respect to the progress of the objectiveenzymatic reaction.

The mechanism of forming the branched α-glucan, when theα-glucosyltransferase of the present invention is allowed to act on anaqueous solution of starch, partial starch hydrolyzate, or amylose, isestimated as follows:

(1) The enzyme acts on maltose and/or α-1,4 glucan having a glucosepolymerization degree of 3 or higher as the substrate and forms theα-1,4 glucan in which a glucose residue is bound via α-linkage tohydroxyl group at C-4 or C-6 position of the non-reducing end glucoseresidue (α-glucan whose glucose polymerization degree is increased byone) and the α-1,4 glucan whose glucose polymerization degree isdecreased by one, by mainly transferring the non-reducing end glucoseresidue to the non-reducing end glucose residue of the other α-1,4glucan by α-1,4 or α-1,6 transglucosylation.(2) The enzyme further acts on the α-1,4 glucan whose glucosepolymerization degree is decreased by one, formed in the above step (1);and transfers a glucose residue to the C-4 or C-6 hydroxyl group of thenon-reducing end glucose residue of the α-glucan whose glucosepolymerization degree is increased by one, also formed in the above step(1), by the intermolecular α-1,4 or α-1,6 transglucosylation to elongatethe glucose chain.(3) By repeating the reactions in the above steps (1) and (2), theenzyme forms a glucan having both α-1,4 and α-1,6 linkages from maltoseand/or α-glucan having a glucose polymerization degree of 3 or higher.(4) Although the frequency is low, the enzyme forms a glucan havingα-1,3, α-1,4,6, and α-1,3,6 linkages in addition to α-1,4 and α-1,6linkages by catalyzing the α-1,3 transglucosylation and α-1,4 or α-1,3transglucosylation to the internal glucose residues involving α-1,6linkages, in the glucan.(5) As results of repeating the reactions in the above steps (1) to (4),the branched α-glucan of the present invention, in which glucose ismainly bound via α-1,4 and α-1,6 linkages and which has α-1,3, α-1,4,6,and α-1,3,6 linkages in a low frequency, is formed by the enzyme.

It was revealed that Bacillus circulans PP710, FERM. BP-10771, capableof producing the α-glucosyltransferase of the present invention, alsoproduces an amylase together with the α-glucosyltransferase of thepresent invention simultaneously. It was also revealed that the branchedα-glucan with a high WSDF content can be unexpectedly produced when theα-glucosyltransferase and the amylase were allowed to act on maltoseand/or α-1,4 glucan having a glucose polymerization degree of 3 orhigher in combination, in comparison with the case of using theα-glucosyltransferase only.

As an example of such amylase produced by Bacillus criculans PP710, FERMBP-10771, the enzyme having the following physicochemical properties canbe used:

(1) Action

-   -   Catalyzing the hydrolysis of starch and the transfer of glycosyl        group, forming cyclodextrins, and hydrolyzing pullulan to form        panose;

(2) Molecular weight

-   -   58,000±10,000 daltons when determined on SDS-polyacrylamide gel        electrophoresis;

(3) Optimum temperature

-   -   55° C. when reacted at pH 6.0 for 30 min;

(4) Optimum pH

-   -   pH 6 to 7 when reacted at 35° C. for 30 min;

(5) Thermal stability

-   -   Stable up to 40° C. when incubated at pH 6.0 for 60 min;    -   Stable up to 50° C. when incubated at pH 6.0 in the presence of        1 mM Ca²⁺ ion; and

(6) pH Stability

-   -   Stable in the pH range of 6.0 to 8.0 when incubated at 4° C. for        24 hours;

The reason why the WSDF content of the branched α-glucan, obtained frompartial starch hydrolyzate by using the α-glucosyltransferase and theamylase in combination, is higher than that of the branched α-glucan,obtained by using the α-glucosyltransferase only, is suggested that theamylase further transfers glycosyl groups to the branched α-glucanformed by the α-glucosyltransferase and the degree of the branch in theglucan is increased.

When the branched α-glucan is prepared by the enzyme reaction, themolecular weight distribution of the branched α-glucan can beadvantageously controlled by using a well-known amylase in combinationwith the α-glucosyltransferase. Also, the digestibility or the reducingpowder of the branched α-glucan can be advantageously decreased by thecombinational use of the enzymes. For example, the branched α-glucanwith a narrow molecular weight distribution, low viscosity, and highcontent of α-1,3, α-1,6, and α-1,3,6 linkages, which involve the lowdigestibility, can be advantageously prepared by allowing an enzyme,which hydrolyzes internal α-1,4 linkages of starch to newly formnon-reducing end glucose residues, such as α-amylase and CGTase to acton liquefied starch in combination with the α-glucosyltransferase of thepresent invention. Also, a starch-debranching enzyme such as isomalyasecan be used together with the α-glucosyltransferase for narrowing therange of molecular weight distribution and lowering the viscosity. Anon-reducing saccharide-forming enzyme (alias “maltooligosyltrehalosesynthase”, (EC 5.4.99.15), disclosed in Japanese Patent Kokai No.143,876/95, can be used together with the α-glucosyltransferase forlowering the reducing power of the branched α-glucan by partiallyconverting reducing end parts into trehalose structure.

In addition, the branched α-glucan of the present invention can beproduced by the steps of culturing a microorganism capable of producingthe α-glucosyltransferase of the present invention in a culture mediumcomprising maltose and/or α-1,4 glucan having a glucose polymerizationdegree of 3 or higher and collecting the formed branched α-glucan fromthe culture broth.

The reaction mixture, thus obtained by the above reaction, can be usedintact as a branched α-glucan product. Optionally, the branched α-glucanhaving low digestibility can be prepared by the steps of hydrolyzing thedigestive parts in the glucan by allowing one or more enzymes selectedfrom the group consisting of α-amylase, β-amylase, glucoamylase, andα-glucosidase to act on the reaction mixture, collecting the resultingnon-digestive fraction by separating methods, and eliminating theresulting hydrolyzates such as glucose by a fermenting treatment usingyeast. Usually, the reaction mixture comprising the branched α-glucan isused after purification. Conventional methods used for purifyingsaccharides can be arbitrarily selected as the purification method. Forexample, one or more purification methods selected from the groupconsisting of decoloring with an activated charcoal; desalting with ionexchange resins in H- and OH-form; separation using organic solventssuch as alcohol and acetone; and separation using a membrane having asuitable separability; can be arbitrarily used.

The α-glucosyltransferase of the present invention hardly produces lowmolecular weight oligosaccharides such as glucose and maltose whenallowed to act on gelatinized starch or partial starch hydrolyzate witha relatively low DE (Dextrose Equivalent), preferably, DE less than 20.Therefore, it is not necessary to purify the reaction product by columnchromatography. However, the reaction product can be arbitrary purifiedfor any purpose. When ion-exchange chromatography is used for purifyingthe branched α-glucan, column chromatography using a strongly acidiccation exchange resin, described in Japanese Patent Kokai Nos. 23,799/83and 72, 598/83, can be advantageously used. In this case, any one offixed bed, moving bed, and semi-moving bed methods can be employed.

The solution containing the branched α-glucan of the present inventionthus obtained can be used intact. However, it is preferable to make thebranched α-glucan into powdery form by drying for preservation andhandling. Usually, various methods such as freeze-drying, spray-drying,and drum drying can be used for drying. Optionally, the dried branchedα-glucan can be arbitrary made into powder with a specific particle sizeby pulverizing, sieving, and granulating.

The branched α-glucan of the present invention exhibits variousproperties such as osmotic pressure-controlling property, excipientproperty, gloss-imparting property, moisture-retaining property,viscosity-imparting property, adhesion property,crystallization-inhibiting property for other saccharides, lowfermentative property, etc. Thus, the branched α-glucan of the presentinvention and the saccharide compositions comprising the same can beadvantageously used as WSDF, quality-improving agent, stabilizer,excipient, etc., for various compositions such as foods and beverages,favorite products, feeds, baits, cosmetics, and pharmaceuticals.

The branched α-glucan of the present invention can be used incombination with other sweeteners, for example, powdery syrup, glucose,fructose, isomerized sugar, sucrose, maltose, trehalose, honey, maplesugar, sorbitol, maltitol, dihydrochalcone, stevioside, α-glycosylstevioside, sweetener of Momordica grosvenori, glycyrrhizin, thaumatin,sucralose, L-aspartyl L-phenylalanine methyl ester, saccharine, glycineand alanine; and fillers such as dextrin, starch, dextran, and lactose.

Further, powdery products of the branched α-glucan of the presentinvention can be arbitrarily used intact or, if necessary, after mixingwith fillers, excipients, binders, etc., and then shaped into variousshapes such as granules, spheres, sticks, plates, cubes, etc.

Since the branched α-glucan of the present invention is hardlydigestible when it is ingested orally, it can be advantageously used asWSDF for general food products. For example, it can be advantageouslyused as a quality-improving agent for various seasonings such as a soysauce, powdered soy sauce, miso, “funmatsu-miso” (a powdered miso),“moromi” (a refined sake), “hishio” (a refined soy sauce), “furikake” (aseasoned fish meal), mayonnaise, dressing, vinegar, “sanbai-zu” (a sauceof sugar, soy sauce and vinegar), “funmatsu-sushi-zu” (powdered vinegarfor sushi), “chuka-no-moto” (an instant mix for Chinese dish),“tentsuyu” (a sauce for Japanese deep fat fried food), “mentsuyu” (asauce for Japanese vermicelli), sauce, catsup, “yakiniku-no-tare” (asauce for Japanese grilled meat), curry roux, instant stew mix, instantsoup mix, “dashi-no-moto” (an instant stock mix), mixed seasoning,“mirin” (a sweet sake), “shin-mirin” (a synthetic mirin), table sugar,and coffee sugar. Also, the branched α-glucan can be advantageously usedto as WSDF, which can be incorporate into various “wagashi” (Japanesecakes) such as “senbei” (a rice cracker), “arare” (a rice cake cube),“okoshi” (a millet and rice cake), “gyuhi” (a starch paste), “mochi” (arise paste) and the like, “manju” (a bun with a bean-jam), “uiro” (asweet rice jelly), “an” (a bean-jam) and the like, “yokan” (a sweetjelly of beans), “mizu-yokan” (a soft azuki-bean jelly), “kingyoku” (akind of yokan), jelly, pao de Castella, and “amedama” (a Japanesetoffee); Western confectioneries such as a bun, biscuit, cracker,cookie, pie, pudding, butter cream, custard cream, cream puff, waffle,sponge cake, doughnut, chocolate, chewing gum, caramel, nougat, andcandy; frozen desserts such as an ice cream and sherbet; syrups such asa “kajitsu-no-syrup-zuke” (a preserved fruit) and “korimitsu” (a sugarsyrup for shaved ice); pastes such as a flour paste, peanut paste, andfruit paste; processed fruits and vegetables such as a jam, marmalade,“syrup-zuke” (fruit pickles), and “toka” (conserves); pickles andpickled products such as a “fukujin-zuke” (red colored radish pickles),“bettara-zuke” (a kind of whole fresh radish pickles), “senmai-zuke” (akind of sliced fresh radish pickles), and “rakkyo-zuke” (pickledshallots); premix for pickles and pickled products such as a“takuan-zuke-no-moto” (a premix for pickled radish), and“hakusai-zuke-no-moto” (a premix for fresh white rape pickles); meatproducts such as a ham and sausage; products of fish meat such as a fishham, fish sausage, “kamaboko” (a steamed fish paste), “chikuwa” (a kindof fish paste), and “tenpura” (a Japanese deep-fat fried fish paste);“chinmi” (relish) such as a “uni-no-shiokara” (salted guts of urchin),“ika-no-shiokara” (salted guts of squid), “su-konbu” (processed tangle),“saki-surume” (dried squid strips), “fugu-no-mirin-boshi” (a driedmirin-seasoned swellfish), seasoned fish flour such as of Pacific cod,sea bream, shrimp, etc.; “tsukudani” (foods boiled down in soy sauce)such as those of laver, edible wild plants, dried squid, small fish, andshellfish; daily dishes such as a “nimame” (cooked beans), potato salad,and “konbu-maki” (a tangle roll); milk products; canned and bottledproducts such as those of meat, fish meat, fruit, and vegetable;alcoholic beverages such as a synthetic sake, fermented liquor, sake,fruit liquor, low-malt beer and beer; soft drinks such as a coffee,cocoa, juice, carbonated beverage, sour milk beverage, and beveragecontaining a lactic acid bacterium; instant food products such asinstant pudding mix, instant hot cake mix, instant juice, instantcoffee, “sokuseki-shiruko” (an instant mix of azuki-bean soup with ricecake), and instant soup mix; and other foods and beverages such as solidfoods for babies, foods for therapy, drinks, peptide foods, and frozenfoods.

The branched α-glucan can be arbitrarily used as feeds and pet foods forimproving the functions of the intestine, improving constipation,inhibiting obesity of animals and pets such as domestic animals,poultry, honeybees, silk warms, and fishes. Also, the branched α-glucancan be advantageously used as a quality-improving agent and stabilizerfor various compositions including favorite products, cosmetics, andpharmaceuticals in a paste or liquid form such as tobacco, cigarette,tooth paste, lipstick, rouge, lip cream, internal liquid medicine,tablet, troche, cod-liver oil in the form of drop, oral refrigerant,cachou, and gargle.

When used as a quality-improving agent or stabilizer, the branchedα-glucan can be advantageously used in biologically active substancessusceptible to lose their effective ingredients and activities, as wellas in health foods, functional foods, and pharmaceuticals containing thebiologically active substances. Example of such biologically activesubstances are liquid preparations containing lymphokines such as α-,β-, and γ-interferons, tumor necrosis factor-α (TNF-α), tumor necrosisfactor-β (TNF-β), macropharge migration inhibitory factor,colony-stimulating factor, transfer factor, and interleukin 2; liquidpreparations containing hormones such as insulin, growth hormone,prolactin, erythropoietin, and follicle-stimulating hormone; liquidbiological preparations such as BCG vaccine, Japanese encephalitisvaccine, measles vaccine, live polio vaccine, small pox vaccine, tetanustoxoid, Trimeresurus antitoxin, and human immunoglobulin; liquidpreparations containing antibiotics such as penicillin, erythromycin,chloramphenicol, tetracycline, streptomycin, and kanamycin sulfate;liquid preparations containing vitamins such as thiamin, riboflavin,L-ascorbic acid, cod liver oil, carotenoid, ergosterol, tocopherol;highly unsaturated fatty acids and their derivatives such as EPA, DHAand arachidonic acid; solution of enzymes such as lipase, esterase,urokinase, protease, β-amylase, isoamylase, glucanase, and lactase;extracts such as ginseng extract, turtle extract, chlorella extract,aloe extract and propolis extract; biologically active substances suchas living microorganisms paste of virus, lactic acid bacteria, andyeast, and royal jelly. By using the branched α-glucan of the presentinvention as a quality-improving agent or stablizer, the abovebiologically active substances can be arbitrary prepared in healthfoods, functional foods, and pharmaceuticals in a liquid, paste, orsolid form, which have a satisfactorily-high stability and quality withless fear of losing or inactivating their effective ingredients andactivities.

The methods for incorporating the branched α-glucan of the presentinvention into the aforesaid compositions are those which canincorporate it before completion of their processing, and which can beappropriately selected from the following conventional methods; mixing,kneading, dissolving, melting, soaking, penetrating, dispersing,applying, coating, spraying, injecting, crystallizing, and solidifying.The amount of the branched α-glucan to be preferably incorporated intothe final compositions is usually in an amount of 0.1% or higher,desirably, 1% or higher.

Further, since the α-glucosyltransferase of the present inventionconverts α-1,4 glucan into the branched α-glucan of the presentinvention when the enzyme is allowed to act on a composition comprisingmaltose and/or α-1,4 glucan having a glucose polymerization degree of 3or higher, the enzyme can be used as a quality-improving agent for thecomposition comprising maltose and/or α-1,4 glucan having a glucosepolymerization degree of 3 or higher.

Furthermore, the α-glucosyltransferase of the present invention convertsmaltose and/or α-1,4 glucan having a glucose polymerization degree of 3or higher into the branched α-glucan of the present invention, and while25% (w/w) or higher but 50% (w/w) or lower, on a dry substrate basis, ofisomaltose is formed by hydrolyzing the branched α-glucan of the presentinvention by using isomaltodextranase (EC 3.2.1.94). Accordingly,isomaltose or a saccharide composition comprising the same can beproduced from maltose and/or α-1,4 glucan having a glucosepolymerization degree of 3 or higher as material by the two-step enzymereactions using the α-glucosyltransferase of the present invention andisomaltodextranase.

The following experiments explain the present invention in detail.

Experiment 1 Preparation of a glucan using α-glucosyltransferase fromBacillus circulans PP710 (FERM BP-10771) Experiment 1-1 Preparation ofα-glucosyltransferase from Bacillus circulans PP710 (FERM BP-10771)

A liquid culture medium consisting of 1.5% (w/v) of “PINEDEX® #4”, apartial starch hydrolyzate commercialized by Matsutani ChemicalIndustries Co., Ltd., Hyogo, Japan, 0.5% (w/v) of “POLYPEPTONE®”, ayeast extract commercialized by Nihon Pharmaceutical Co., Ltd., Tokyo,Japan, 0.1% (w/v) of “YEAST EXTRACT S”, a yeast extract commercializedby Nihon Pharmaceutical Co., Ltd., Tokyo, Japan, 0.1% (w/v) ofdipotassium phosphate, 0.06% (w/v) of sodium phosphate dihydrate, 0.05%(w/v) of magnesium sulfate hepta-hydrate, 0.001% (w/v) of manganesesulfate penta-hydrate, 0.001%(w/v) of ferrous sulfate hepta-hydrate, andwater was placed in a 500 ml-Erlenmeyer flask in an amount of 100 ml,sterilized by autoclaving at 121° C. for 20 min, and cooled.Successively, the culture medium was inoculated with Bacillus circulansPP710, FERM BP-10771, and followed by cultivation under rotary-shakingconditions at 27° C. and 230 rpm for 48 hours to obtain a seed culture.

A fresh preparation of the same culture medium was placed in twelve 500ml-Erlenmeyer flasks in respective amounts of 100 ml, sterilized byheating and cooled to 27° C. Successively, one milliliter each of theabove seed culture was inoculated to the medium and followed bycultivation under rotary-shaking conditions at 27° C. for 24 hours.After completion of the culture, the culture broth was withdrawn fromeach of the Erlenmeyer flasks and centrifuged at 8,000 rpm for 20minutes to remove cells. The α-glucosyltransferase activity of theresulting culture supernatant was assayed and determined to be 2.8units/ml. About one liter of the culture supernatant was salted out byadding ammonium sulfate to give finally 80% saturation and allowing itto stand at 4° C. for 24 hours. The resultant precipitates werecollected by centrifuging at 11,000 rpm for 30 min, dissolved in 20 mMacetate buffer (pH 4.5), and dialyzed against a fresh preparation of thesame buffer to obtain about 20 ml of a crude enzyme solution. The crudeenzyme solution was subjected to cation-exchange column chromatographyusing 20 ml of “CM-TOYOPEARL™ 650S” gel, a cation-exchange gelcommercialized by Tosoh Corporation, Tokyo, Japan, pre-equilibrated with20 mM acetate buffer (pH 4.5). After eluting non-absorbed proteins, theactive fractions were eluted by a linear gradient of 0 to 0.5 M sodiumchloride. The active fractions, eluted at about 0.18 to 0.45 M sodiumchloride, were collected and dialyzed against 20 mM acetate buffer (pH6.0). The resulting dialyzate was used as a preparation ofα-glucosyltransferase.

Experiment 1-2 Preparation of a branched α-glucan usingα-glucosyltransferase

One hundred milliliter of the preparation of α-glucosyltransferase,obtained in Experiment 1-1, was used as an enzyme solution. “PINEDEX®#100”, a partial starch hydrolyzate commercialized by Matsutani ChemicalIndustries Co., Ltd., Hyogo, Japan, was admixed with the enzyme solutionto give a final concentration of 30% (w/v), followed by the enzymereaction at 40° C. for 72 hours, and then heated at about 100° C. for 10minutes to stop the reaction. After removing the resultant insolublesubstances by filtration, the filtrate was decolored and desalted using“DIAION™ SK-1B” and “DIAION™ WA30”, ion exchange resins commercializedby Mitsubishi Chemical Corporation, Tokyo, Japan, and “IRA 411”, ananion exchange resin commercialized by Organo Corporation, Tokyo, Japan.The resulting solution was filtrated and concentrated using anevaporator, and a 30% (w/w) glucan solution was obtained in a yield of85.8%, on a dry solid basis, from the partial starch hydrolyzate used assubstrate.

Experiment 2 Preparation of a Glucan Using α-Glucosyltransferase fromArthrobacter Globiformis PP349 (FERM BP-10770) Experiment 2-1Preparation of α-Glucosyltransferase from Arthrobacter globiformis PP349(FERM BP-10770)

Except for inoculating Arthrobacter globiformis PP349, FERM BP-10770,instead of Bacillus circulans PP710, FERM BP-10771, a seed culture wasprepared according to the method in Experiment 1-1.

A fresh preparation of the same culture medium used for the seed culturewas placed in twelve 500 ml-Erlenmeyer flasks in respective amounts of100 ml, sterilized by heating and cooled to 27° C. Successively, onemilliliter each of the above seed culture was inoculated and followed bycultivation under rotary-shaking conditions at 27° C. for 24 hours.After completion of the culture, the culture broth was withdrawn fromeach of Erlenmeyer flasks and centrifuged at 8,000 rpm for 20 minutes toremove cells. The α-glucosyltransferase activity of the resultingculture supernatant was assayed and determined to be 0.53 unit/ml. Aboutone liter of the culture supernatant was salted out by adding ammoniumsulfate to give finally 80% saturation and allowing it to stand at 4° C.for 24 hours. The resultant precipitates were collected by centrifugingat 11,000 rpm for 30 min, dissolved in 20 mM acetate buffer (pH 6.0),and dialyzed against the same buffer to obtain about 20 ml of a crudeenzyme solution. The crude enzyme solution was subjected toanion-exchange column chromatography using 20 ml of “DEAF-TOYOPEARL™650S” gel, an anion-exchange gel commercialized by Tosoh Corporation,Tokyo, Japan, pre-equilibrated with 20 mM acetate buffer (pH 6.0). Aftereluting non-absorbed proteins, the active fractions were eluted by alinear gradient of zero to 0.5 M sodium chloride. The active fractions,eluted at about 0.05 to 0.2 M sodium chloride, were collected anddialyzed against 20 mM acetate buffer (pH 6.0). The resulting dialyzatewas used as a preparation of α-glucosyltransferase.

Experiment 2-2 Preparation of a Branched α-Glucan Usingα-Glucosyltransferase

One hundred milliliter of the preparation of α-glucosyltransferase,obtained in Experiment 2-1, was used as an enzyme solution. “PINEDEX®#100”, a partial starch hydrolyzate commercialized by Matsutani ChemicalIndustries Co., Ltd., Hyogo, Japan, was admixed with the enzyme solutionto give a final concentration of 30% (w/v), followed by the enzymereaction at 40° C. for 72 hours, and then heated at about 100° C. for 10minutes to stop the reaction. After removing the resultant insolublesubstances by filtration, the filtrate was decolored and desalted using“DIAION™ SK-1B” and “DIAION™ WA30”, ion exchange resins commercializedby Mitsubishi Chemical Corporation, Tokyo, Japan, and “IRA 411”, ananion exchange resin commercialized by Organo Corporation, Tokyo, Japan.The resulting solution was filtrated and concentrated using aevaporator, and a 30% (w/w) glucan solution was obtained in a yield of83.6%, on a dry solid basis, from the partial starch hydrolyzate used assubstrate.

In the following Experiments 3 and 4, the glucans, obtained inExperiments 1-2 and 2-2, were called to “Glucan A” and “Glucan B”,respectively, for the distinction.

Experiment 3 Evaluation of Glucan A and B as WSDF

According to the method described in Section 8, Dietary fiber, (2)High-performance liquid chromatography (Enzyme-HPLC method), “Methodsfor analyzing nutritional components (Appendix 1-3 of Nutrition LabelingStandard) in Nutrition Labeling Standard (Notification No. 146 ofMinistry of Health, Labour, and Welfare, May, 1986)”, the WSDF contentsof Glucans A and B were determined as follows: “DIETARY FIBER, TOTALASSAY, CONTROL KIT”, a kit for determining total amount of dietaryfiber, commercialized by Sigma-Aldrich Japan, was used as a kit forenzymatic treatments. “PINEDEX® #100”, a partial starch hydrolyzatecommercialized by Matsutani Chemical Industries Co., Ltd., Hyogo, Japan,used for preparing Glucans A and B as substrate, was used as Control 1.“PINEFIBER®”, a commercially available low-digestible glucancommercialized by Matsutani Chemical Industries Co., Ltd., Hyogo, Japan,was used as Control 2.

<Preparation of Sample Solution for Analyses>

To a test tube, 0.1 g-dry solid of each glucan was sampled and thenadmixed with 5 ml of 0.08 M sodium phosphate buffer to adjust the pH to6.0. Successively, 0.01 ml of a thermostable α-amylase (which is derivedfrom Bacillus licheniformis and commercialized by Sigma-Aldrich Japan)solution, attached to the kit, was admixed with the above solution andthen the tube containing the mixture was wrapped with aluminum foil, andfollowed by the enzyme reaction in a boiling water bath with stirring at5 min-interval for 30 min. After the reaction, the reaction mixture wascooled and adjusted the pH to 7.5 by adding 1 ml of 0.275 M sodiumhydroxide solution. The resulting solution was admixed with 0.01 ml of aprotease (which is derived from Bacillus licheniformis andcommercialized by Sigma-Aldrich Japan) solution, attached to the kit,and then the tube containing the mixture was wrapped with aluminum foil,and followed by the enzyme reaction in a water bath with shaking at 60°C. for 30 min, and then cooled. After adjusting the pH of the resultingsolution after the protease treatment to 4.3 by adding about 1 ml of0.325 M hydrochloric acid solution, the resulting solution was furtheradmixed with 0.01 ml of the amyloglucosidase (which is derived fromAspergillus niger and commercialized by Sigma-Aldrich Japan) solution,attached to the kit, and then the tube containing the mixture waswrapped with aluminum foil, and followed by the enzyme reaction in awater bath with shaking at 60° C. for 30 min, and then cooled.Successively, about 7 ml of the resulting reaction mixture was subjectedto an ion-exchange column, which is prepared by mixing “AMBERLITE™IRA-67” (OH-form) and “AMBERLITE™ 200CT” (H-form), both commercializedby Organo Corporation, Tokyo, Japan, in a ratio of 1:1, eluted at SV 1.0for desalting, and further eluted with about 3-folds volume of deionizedwater, and then filled up to the total volume of about 28 ml. The elutewas concentrated using an evaporator, filtrated using a membrane filterwith a pore size of 0.45 μm, and then filled up to 25 ml to make into asample solution for the analysis.

<Conditions for High-Performance Liquid Chromatography>

The test sample solution, obtained by the method described above, wassubjected to a high performance liquid chromatography under thefollowing conditions:

-   -   Column: “TSK Gel™ G2500PWXL” (ID 7.8 mm×length 300 mm), produced        by Tosoh Corporation, Tokyo, Japan; two columns were connected        in series    -   Eluent: Deionized water    -   Saccharide concentration of test sample: 0.8% (w/w)    -   Column temperature: 80° C.    -   Flow rate: 0.5 ml/min    -   Detector: Refractive index detector    -   Injection: 20 μl    -   Time for analysis: 50 min

<Calculation of the Dietary Fiber Content in the Test Samples>

In the chromatogram obtained by the above HPLC, undigested glucan whichremained after the enzyme treatments was assumed to WSDF. The peak areasof the WSDF and glucose formed by the digestion were measured,respectively. Separately, the amount of glucose in the test sample wasdetermined by conventional glucose oxidase-peroxidase method. Using thevalues of the above peak areas and the amount of glucose, the amount ofthe WSDF was calculated by the following Formula 1. Then, the WSDFcontent in the test sample was calculated by the following Formula 2.

The amount of WSDF*(mg)={(The peak area of WSDF)/(The peak area ofglucose)}×(The amount of glucose in the test samplesolution)**(mg)  Formula 1

*: Water-soluble dietary fiber**: Concentration of glucose in the test sample solution (mg/ml)×25 ml

The WSDF content(%,w/w)={(The amount of WSDF in the testsample)(mg)/(The amount of the test sample)(mg)}×100  Formula 2

The WSDF contents of Glucans A and B, calculated by the aboveEnzyme-HPLC method, were 42.1% (w/w) and 41.8% (w/w), respectively.While, in the case of the partial starch hydrolyzate, Control 1, it wascompletely hydrolyzed into glucose by the enzyme treatments, and theWSDF content of the partial starch hydrolyzate was estimated to be zero% (w/w). That of “PINEFIBER®”, a low-digestible dextrin commercializedby Matsutani Chemical Industries Co., Ltd., Hyogo, Japan, Control 2, wasestimated to be 48.7% (w/w). These results indicate that a glucan whichshows the almost equal WSDF content with a commercial low-digestibledextrin can be easily prepared by allowing the α-glucosyltransferase ofthe present invention to act on a partial starch hydrolyzate, notcomprising WSDF, as a substrate.

Experiment 4 Structural Analyses of Glucans A and B Experiment 4-1Methylation Analysis

According to conventional method, Glucans A and B, respectively obtainedin Experiments 1-2 and 2-2, were subjected to the methylation analyses,and the resulting partially methylated products were subjected to thegas chromatography under the following conditions, and the results arein Table 3:

<Conditions for Gas Chromatography>

-   -   Column: “DB-15” (ID 0.25 mm×length 30 m, film thickness 1 μm), a        capillary column produced by J&W Scientific, Tokyo, Japan;    -   Carrier gas: Helium    -   Column temperature: kept at 130° C. for 2 min, heated to 250° C.        in a rate of 5° C./min, and then kept at 250° C. for 20 min    -   Flow rate: 1.0 ml/min    -   Detector: FID    -   Injection: 3 μl (split: 1/30)    -   Time for analysis: 46 min

TABLE 3 Composition (Peak area %) Partial starch hydrolyzate* Partiallymethylated product Corresponding Glc** (Material) Glucan A Glucan B2,3,4,6-Tetramethylated product Non-reducing end Glc 5.8 10.1 9.83,4,6-Trimethylated product Glc involving 1,2-linkage 0.0 0.0 0.02,4,6-Trimethylated product Glc involving 1,3-linkage 0.0 1.1 0.92,3,6-Trimethylated product Glc involving 1,4-linkage 89.8 51.5 49.12,3,4-Trimethylated product Glc involving 1,6-linkage 0.0 32.2 33.92,4-Dimethylated product Glc involving 1,3,6-linkage 0.0 0.8 1.12,3-Dimethylated product Glc involving 1,4,6-linkage 4.4 4.5 5.2*PINEDEX ® #100, a partial starch hydrolyzate commercialized byMatsutani Chemical Industries Co., Ltd. **Glucose residue Glucan A:Glucan prepared by using α-glucosyltransferase from strain PP710 GlucanB: Glucan prepared by using α-glucosyltransferase from strain PP349

As is evident from the results in Table 3, in both cases of Glucans Aand B, 2,3,6-trimethylated product was significantly decreased and2,3,4-trimethylated product was significantly increased to 30% or higherin comparison with the results of methylation analysis of Glucans A andB, which were respectively prepared using α-glucosyltransferase derivedfrom Bacillus cruculans PP710 and Arthrobacter globiformis PP349; andthat of the partial starch hydrolyzate used as substrate. These resultsindicate that the partial starch hydrolyzate having a structure ofpolymerizing glucoses mainly via α-1,4 linkages is converted into thebranched α-glucan having 30% or higher of α-1,6 linkages by the reactionof α-glucosyltrasferase from Bacillus circulans PP710 and Arthrobacterglobiformis PP349. It was also revealed that non-reducing ends, α-1,3linkages and α-1,3,6 linkages were newly formed because2,3,4,6-tetramethylated product, 2,4,6-trimethylated product, and2,4-dimethylated product were also increased. The contents of2,4,6-trimethylated product and 2,4-dimethylated product in thepartiallymethylatedproducts of Glucan A were 1.1% and 0.8%,respectively. The contents of 2,4,6-trimethylated product and2,4-dimethylated product in the partially methylated products of GlucanB were 0.9% and 1.1%, respectively. Further, it was considered that thecontent of α-1,4,6 linkage, which is inherently present in the substrateas a branched point, was not so changed because the contents of2,3-dimethylated product in the partially methylated products fromGlucans A and B were not so different from that from the substrate. Fromthese results, it was revealed that Glucans A and B were a branchedglucan (branched α-glucan) having α-1,4 linkage and α-1,6 linkage asmajor glucosidic linkage and α-1,3 linkage and α-1,3,6 linkage as minorglucosidic linkage different from the partial starch hydrolyzate used assubstrate. It was also revealed from their ¹H-NMR spectra obtained byNMR analyses that all amomeric form of C-1 position of glucose,constituting Glucans A and B, were α-form.

Experiment 4-2 Isomaltodextranase Digestion of Branched α-Glucan A and B

In order to characterize the structures of Branched α-glucans A and B,isomaltodextranse digestion of them was carried out. An aqueous solutionof Branched α-glucan A or B with a final concentration of 1% (w/v) wasadmixed with 100 units/g-solid substrate of isomaltodextranase, derivedfrom Arthrobacter globiformis and prepared in Hayashibara BiochemicalLaboratories Inc., Okayama, Japan; and followed the enzyme reaction atpH 5.0 and 50° C. for 16 hours. After stopping the reaction by keepingat 100° C. for 10 min, the saccharide composition in the resultingreaction mixture was determined using high-performance liquidchromatography (hereinafter, abbreviated as “HPLC”) and gaschromatography (hereinafter, abbreviated as “GC”). HPLC was carried outunder the following conditions:

<Conditions for HPLC>

-   -   Column: “MCI GEL CKO4SS”, produced by Mitsubishi Chemical        Corporation, Tokyo, Japan; two columns were connected in series    -   Eluent: Water    -   Column temperature: 80° C.    -   Flow rate: 0.4 ml/min    -   Detector: “RID-10A”, a refractive index detector produced by        Shimadzu Corporation, Kyoto, Japan.

GC was carried out after converting saccharides intotrimethylsiliyl-derivatives (TMS-derivatives) and under the followingconditions:

<Conditions for GC>

-   -   Column: “2% Silicon OV-17 Chromosorb W/AW-DMS”, produced by GL        Science, Tokyo, Japan;    -   Column temperature: raised 160° C. to 320° C. in a rate of 7.5°        C./min    -   Carrier gas: Nitrogen    -   Detector: FID.

By the isomaltodextranase digestion, no isomaltose was formed from thepartial starch hydrolyzate, the substrate used for preparing thebranched α-glucan. On the contrary, isomaltose in amounts of 28.4% (w/w)and 27.2% (w/w) were formed from Branched α-glucan A and B,respectively, by the digestion. These results indicate that Branchedα-glucan A and B have isomaltose structure in amounts of about 28.4%(w/w) and 27.2% (w/w). Also, they support the results of the methylationanalyses in Experiment 4-1, showing that the ratio of α-1,6 linkage isincreased in the branched α-glucan. Since isomaltodextranase has aspecificity of hydrolyzing α-glucosidic linkage adjacent to the reducingend of isomaltose structure in glucan with no distinction of α-1,3,α-1,4, and α-1,6 linkages, it is not clear in detail how the resultingisomaltose are bound via any one of the above linkages.

Experiment 4-2 α-Glucosidase/Glucoamylase Double Digestion of Branchedα-Glucan A and B

α-Glucosidase/glucoamylase double digestion test of Branched α-glucan Aor B was carried out by allowing “TRANSGLUCOSIDASE AMANO L”,α-glucosidase from Aspergillus niger and glucoamylase from Rhizopus sp.to act simultaneously on Branched α-glucan A or B. The aqueous solutioncontaining Branched α-glucan A or B was admixed with 5,000units/g-solid-substrate of α-glucosidase and 100 units/g-solid-substrateof glucoamylase and followed by the enzyme reaction at pH 5.5 and 50° C.for 16 hours. After stopping the reaction by keeping the reactionmixture at 100° C. for 10 min, the saccharide composition of thereaction mixture was analyzed by HPLC under the same condition inExperiment 4-2. As a result, both Branched α-glucan A and B weresubstantially hydrolyzed into glucose as in the case of the partialstarch hydrolyzate used as substrate for preparing the branchedα-glucans. These results indicate that both Branched α-glucan A and Bare α-glucan constructed by glucose molecules as component sugar.

Experiment 4-4 Analysis of the Molecular Weight Distribution

The molecular weight distributions of Branched α-glucans A and B wereanalyzed by the conventional gel-filtration HPLC. The gel-filtrationHPLC was carried out by the following conditions:

<Conditions for Gel-Filtrating HPLC>

-   -   Column: “TSK GEL α-M”, produced by Tosoh Corporation, Tokyo,        Japan; two columns were connected in series    -   Eluent: 10 mM sodium phosphate buffer (pH 7.0)    -   Column temperature: 40° C.    -   Flow rate: 0.3 ml/min    -   Detector: “RID-10A”, a refractive index detector produced by        Shimadzu Corporation, Kyoto, Japan.

The molecular weight of glucans in the samples was calculated based onthe molecular weight-calibration curve prepared by subjecting “Standardpullulan for measuring molecular weight”, commercialized by HayashibaraBiochemical Laboratories Inc., Okayama, Japan, to the same gelfiltration HPLC analysis. FIG. 1 shows a comparative gel-filtration HPLCchromatograms of Branched α-glucans A and B (Symbols “b” and “c” in FIG.1), and “PINEDEX® #100”, the partial starch hydrolyzate (Symbol “a” inFIG. 1) used as substrate for preparing Branched α-glucan A and B. InFIG. 1, symbols “A”, “B”, “C”, “D”, and “E” mean the positions ofeluting glucan having a molecular weight of 1,000,000, 100,000, 10,000,1,000, and 100 daltons, respectively. (Also in the cases of FIGS. 15 and19 mentioned after) The results of the molecular weight distributionanalyses of samples based on the gel-filtration HPLC chromatograms arein Table 4.

TABLE 4 Partial starch hydrolyzate* Analytical data (Material) Glucan AGlucan B Number average molecular 6,680 3,840 4,050 weight (Mn) (Dalton)Weight-average molecular 98,890 59,000 65,700 weight (Mw) (Dalton) Mw/Mn14.8 15.4 16.2 Average glucose 449 and 6.3 384, 22.2, 433, 22.8,polymerization degree 10.9, and 1 10.9, and 1 of peaks *PINEDEX ® #100,a partial starch hydrolyzate commercialized by Matsutani ChemicalIndustries Co., Ltd., Hyogo, Japan Glucan A: Glucan prepared by usingα-glucosyltransferase from strain PP710 Glucan B: Glucan prepared byusing α-glucosyltransferase from strain PP349

In the molecular weight distribution analysis, the partial starchhydrolyzate used as substrate was characterized as a saccharide mixtureshowing two peaks (Symbols “1” and “2” in the chromatogram “a” inFIG. 1) corresponding to the glucose polymerization degree of 499 and6.3. While, Branched α-glucan A was characterized as a saccharidemixture showing four peaks (Symbols “3”, “4”, “5”, and “6” in thechromatogram “b” in FIG. 1) corresponding to the glucose polymerizationdegree of 384, 22.2, 10.9, and 1. Branched α-glucan B was characterizedas a saccharide mixture showing four peaks (Symbols “7”, “8”, “9”, and“10” in the chromatogram “c” in FIG. 1) corresponding to the glucosepolymerization degree of 433, 22.8, 10.9, and 1. The peaks of symbol “6”and “10” correspond to glucose and the facts that the amounts of glucoseare low indicate that the enzyme from Bacillus criculans PP710 andArthrobacter globiformis PP349 has a relatively weak hydrolyticactivity. As is evident from Table 4, both the number average molecularweight and the weight-average molecular weight of Glucan A and B weredecreased to about 60% of those of the partial starch hydrolyzate usedas substrate, and Glucan A and B were converted into molecules with alow-molecular weight as a whole. The value of dividing theweight-average molecular weight with the number average molecular weight(Mw/Mn), which is an index of molecula weigh distribution, was not sochanged among the partial starch hydrolyzate, Glucan A and Glucan B.From the results, it was considered that the both α-glucosyltransferasesfrom Bacillus circulans PP710 and Arthrobacter globiformis PP349specifically act on non-reducing ends of the partial starch hydrolyzate.

From the results in Table 3, it was revealed that, in the partial starchhydrolyzate used as substrate, about 90% of glucosidic linkages areα-1,4 linkages and α-1,4,6 linkages are slightly present in themolecule. On the contrary, it was revealed that, in Glucans A and B, theratio of α-1,6 linkage to α-1,4 linkage is significantly high, andGlucan A and B also have α-1,3 linkages and α-1,3, 6 linkages inaddition to α-1,4,6 linkages. The branched α-glucan having a structureas such has been hitherto unknown.

The structure of the branched α-glucan of the present invention wasdeduced based on the results obtained by the methylation analysis. Thereference diagram of the branched α-glucan is shown in FIG. 2 togetherwith that of the partial starch hydrolyzate used as substrate. In FIG.2, symbols “1” and “2” respectively represent reference diagrams of thepartial starch hydrolyzate used as substrate and the branched α-glucan.Also, in FIG. 2, symbols “a”, “b”, “c”, “d”, “e”, and “f” representnon-reducing end glucose residue, glucose residue involving α-1,3linkage, that involving α-1,4 linkage, that involving α-1,6 linkage,that involving α-1,3,6 linkage, and that involving α-1,4,6 linkage,respectively, in the partial starch hydrolyzate and the branchedα-glucan of the present invention. Further, in the reference diagrams,diagonal broken line, horizontal solid line, and vertical solid line,between glucose residues represent α-1,3 linkage, α-1,4 linkage, andα-1,6 linkage, respectively.

Experiment 5 Preparation of the α-Glucosyltransferase from Bacilluscirculans PP710

A liquid culture medium consisting of 1.5% (w/v) of “PINEDEX® #4”, apartial starch hydrolyzate commercialized by Matsutani ChemicalIndustries Co., Ltd., Hyogo, Japan, 0.5% (w/v) of “POLYPEPTONE”, a yeastextract commercialized by Nihon Pharmaceutical Co., Ltd., Tokyo, Japan,0.1% (w/v) of “YEAST EXTRACT S”, a yeast extract commercialized by NihonPharmaceutical Co., Ltd., Tokyo, Japan, 0.1% (w/v) of dipotassiumphosphate, 0.06% (w/v) of sodium phosphate dihydrate, 0.05% (w/v) ofmagnesium sulfate hepta-hydrate, 0.001% (w/v) of manganese sulfatepenta-hydrate, 0.001% (w/v) of ferrous sulfate hepta-hydrate, and waterwas placed in a 500 ml-Erlenmeyer flask in a amount of 100 ml,sterilized by autoclaving at 121° C. for 20 min, and cooled.Successively, the culture medium was inoculated with Bacillus circulansPP710, FERM BP-10771, and followed by cultivation under rotary-shakingconditions at 27° C. and 230 rpm for 48 hours to obtain a seed culture.

About 20 L of a fresh preparation of the same liquid culture medium asused in the above seed culture were placed in a 30-L fermenter,sterilized by heating, and then cooled to 27° C. and inoculated withabout 200 ml of the seed culture, followed by the cultivation at 27° C.and pH 5.5 to 8.0 for 24 hours under aeration-agitation conditions.After completion of the cultivation, the resulting culture broth wasdistilled from the fermenter and removed cells by centrifuging at 8,000rpm for 20 min, and about 18 L of culture supernatant was obtained. Theα-glucosyltransferase activities in the culture broth and culturesupernatant were assayed. About 2.7 units/ml and about 2.6 units/ml ofthe enzyme activities were detected in the culture broth and the culturesupernatant, respectively. It was revealed that major part of theα-glucosyltransferase of the present invention, produced by Bacilluscirculans PP710, was secreted extracellularly.

Experiment 6 Purification of the α-Glucosyltransferase from Bacilluscirculans PP710

About four liters (Total activity: about 10,400 units) of the culturesupernatant obtained in Experiment 5 was salted out by adding ammoniumsulfate to give finally 80% saturation and allowing it to stand at 4° C.for 24 hours. The resultant precipitates were collected by centrifugingat 11,000 rpm for 30 min, dissolved in 20 mM acetate buffer (pH 4.5),and dialyzed against the same buffer to obtain about 65 ml of a crudeenzyme solution. The crude enzyme solution had about 74 units/ml (Totalactivity: about 4,780 units) of the α-glucosyltransferase. The crudeenzyme solution was subjected to cation-exchange column chromatographyusing 70 ml of “CM-TOYOPEARL™ 650S” gel, a cation-exchange gelcommercialized by Tosoh Corporation, Tokyo, Japan. Theα-glucosyltransferase activity was adsorbed on “CM-TOYOPEARL™ 650S” gelpre-equilibrated with 20 mM acetate buffer (pH 4.5) and eluted at about0.4 M sodium chloride when the elution was carried out with a linergradient of zero to 0.5 M of sodium chloride. The active fractions werecollected and admixed with ammonium sulfate to give a finalconcentration of 1 M, and then allowed to stand at 4° C. for 24 hours.The enzyme solution was centrifuged to remove precipitates, andsubjected to hydrophobic column chromatography using 9 ml of“BUTYL-TOYOPEARL™ 650M” gel, a gel commercialized by Tosoh Corporation,Tokyo, Japan. The α-glucosyltransferase activity was adsorbed on“BUTYL-TOYOPEARL™ 650M” gel pre-equilibrated with 20 mM acetate buffer(pH 6.0) containing 1 M of ammonium sulfate and when eluted with alinear gradient decreasing from 1 M to zero M of ammonium sulfate, theenzyme activity was eluted at about 0.2 M of ammonium sulfate. Theactive fractions were collected, dialyzed against 20 mM acetate buffer(pH 4.5), and subjected to cation-exchange column chromatography using3.3 ml of “CM-5PW™” gel, a gel commercialized by Tosoh Corporation,Tokyo, Japan. The α-glucosyltransferase activity was adsorbed on“CM-5PW™” gel pre-equilibrated with 20 mM acetate buffer (pH 4.5) andwhen eluted with a linear gradient of zero to 1 M of sodium chloride,the enzyme activity was eluted at about 0.4 M of sodium chloride. Theactive fractions were collected and dialyzed against 20 mM acetatebuffer (pH 6.0). The amount of enzyme activity, specific activity, andyield of the α-glucosyltransferase at each purification step are inTable 5.

TABLE 5 α-Glucosyl- Specific activity transferase of α-glucosyl-activity transferase Yield Purification step (units) (units/mg-protein)(%) Culture supernatant 10,400 10 100 Dialyzed solution after 4,780 22.846.0 salting out with ammonium sulfate Eluate from ion-exchange 3,960265 38.1 column chromatography Eluate from hydrophobic 3,800 307 36.5column chromatography Eluate from ion-exchange 3,300 327 31.7 columnchromatography

The finally purified enzyme preparation of the α-glucosyltransferase wasassayed for purity on gel electrophoresis using a 5-20% (w/v) gradientpolyacrylamide gel and detected on the gel as a single protein band,i.e., a high purity preparation.

Experiment 7 Properties of the α-Glucosyltransferase from Bacilluscirculans PP710 Experiment 7-1 Molecular Weight

The purified enzyme preparation of the α-glucosyltransferase, obtainedby the method in Experiment 6, was subjected to SDS-PAGE (a 5 to 20%(w/v) gradient gel) and the molecular weight of the enzyme was measuredcomparing with molecular weight markers, commercialized by Bio-RadJapan, Tokyo, Japan. It was revealed that the α-glucosyltransferase hasa molecular weight of 90,000±10,000 daltons.

Experiment 7-2 Optimum Temperature and Optimum pH for the EnzymeReaction

Effects of temperature and pH on the enzyme activity were investigatedusing the purified enzyme preparation of the α-glucosyltransferase,obtained by the method in Experiment 6, by varying temperature and pH atthe assay of the enzyme. The results are in FIG. 3 (Optimum temperature)and FIG. 4 (Optimum pH), respectively. It was revealed that the optimumtemperature of the α-glucosyltransferase was 50 to 55° C. when reactedat pH 6.0 for 30 min and the optimum pH was 5.0 to 6.3 when reacted at40° C. for 30 min.

Experiment 7-3 Thermal and pH Stabilities of the Enzyme

Thermal stability and pH stability of the enzyme were investigated usingthe purified enzyme preparation of the α-glucosyltransferase, obtainedby the method in Experiment 6. Thermal stability of the enzyme wasdetermined by the steps of incubating an enzyme solution (20 mM acetatebuffer, pH 6.0) under various temperatures for 60 min, cooling in water,and measuring the residual enzyme activity. pH Stability of the enzymewas determined by the steps of incubating enzyme solution in 20 mMbuffer at various pHs, and at 4° C. for 24 hours, adjusting the pH to6.0, and measuring the residual enzyme activity. The results are in FIG.5 (Thermal stability) and in FIG. 6 (pH Stability), respectively. As isevident from the results in FIGS. 5 and 6, the α-glucosyltransferase isstable up to 40° C. and in the range of pH 3.5 to 8.4.

Experiment 7-4 Effects of Metal Ions on the Enzyme Activity

Effects of metal ions on the enzyme activity were investigated using thepurified enzyme preparation of the α-glucosyltransferase, obtained bythe method in Experiment 6, in the presence of 1 mM of respective metalions according to the assay method. The results are in Table 6.

TABLE 6 Metal Relative Metal Relative salt activity (%) salt activity(%) None 100 MgCl₂ 102 CaCl₂ 101 MnCl₂ 99 CoCl₂ 100 NiCl₂ 102 CuCl₂ 52ZnCl₂ 101 FeCl₂ 91 PbCl₂ 97 FeCl₃ 94 EDTA 99 HgCl₂ 3

As is evident from the results in Table 6, it was revealed that theα-glucosyltransferase activity was remarkably inhibited by Hg²⁺ ion andmoderately by Cu²⁺ ion, respectively.

Experiment 8 Preparation of the α-glucosyltransferase from Arthrobacterglobiformis PP394 (FERM BP-10700)

Except for inoculating Arthrobacter globiformis PP349, FERM BP-10770,instead of Bacillus circulans PP710, FERM BP-10771, a seed culture wasprepared according to the method in Experiment 1-1.

About 20 L of a fresh preparation of the same liquid culture medium asused in the above seed culture were placed in a 30-L fermenter,sterilized by heating, and then cooled to 27° C. and inoculated withabout 200 ml of the seed culture, followed by the cultivation at 27° C.and pH 5.5 to 7.0 for 24 hours under aeration-agitation conditions.After completion of the cultivation, the resulting culture broth wasdistilled from the fermenter and removed cells by centrifuging at 8,000rpm for 20 min, and about 18 L of culture supernatant was obtained. Theα-glucosyltransferase activities in the culture broth and culturesupernatant were assayed. About 0.36 unit/ml and about 0.42 unit/ml ofthe enzyme activities were detected in the culture broth and the culturesupernatant, respectively. It was revealed that major part of theα-glucosyltransferase, produced by Arthrobacter globiformis PP349, wassecreted extracellularly.

Experiment 9 Purification of the α-Glucosyltransferase from Arthrobacterglobiformis PP394

About 18 liters (Total activity: about 7,560 units) of the culturesupernatant obtained in Experiment 8 was salted out by adding ammoniumsulfate to give finally 80% saturation and allowing it to stand at 4° C.for 24 hours. The resultant precipitates were collected by centrifugingat 11,000 rpm for 30 min, dissolved in 20 mM acetate buffer (pH 6.0),and dialyzed against the same buffer to obtain about 500 ml of a crudeenzyme solution. The crude enzyme solution had about 14 units/ml (Totalactivity: about 7,000 units) of the α-glucosyltransferase. The crudeenzyme solution was admixed with ammonium sulfate to give a finalconcentration of 2 M, centrifuged to remove precipitates, and thensubjected to hydrophobic column chromatography using 300 ml of“PHENYL-TOYOPEARL™ 650M” gel, a gel commercialized by Tosoh Corporation,Tokyo, Japan. The α-glucosyltransferase activity was adsorbed on“PHENYL-TOYOPEARL™ 650M” gel pre-equilibrated with 20 mM acetate buffer(pH 6.0) containing 2 M of ammonium sulfate and when eluted with alinear gradient decreasing from 2 M to zero M of ammonium sulfate, theenzyme activity was eluted at about 0.6 M of ammonium sulfate. Theactive fractions were collected, dialyzed against 20 mM acetate buffer(pH 6.0), and subjected to anion-exchange column chromatography using100 ml of “DEAF-TOYOPEARL™ 650S” gel, a gel commercialized by TosohCorporation, Tokyo, Japan. The α-glucosyltransferase activity wasadsorbed on “DEAF-TOYOPEARL™ 650S” gel pre-equilibrated with 20 mMacetate buffer (pH 6.0) and when eluted with a linear gradient of zeroto 0.5 M of sodium chloride, the enzyme activity was eluted at about 0.1M of sodium chloride. The amount of enzyme activity, specific activity,and yield of the α-glucosyltransferase at each purification step are inTable 7.

TABLE 7 α-Glucosyl- Specific activity transferase of α-glucosyl-activity transferase Yield Purification step (units) (units/mg-protein)(%) Culture supernatant 7,560 1.6 100 Dialyzed solution after 7,000 5.492.6 salting out with ammonium sulfate Eluate from hydrophobic 3,870 13054.2 column chromatography Eluate from ion-exchange 2,710 415 35.8column chromatography

The finally purified enzyme preparation of the α-glucosyltransferase wasassayed for purity on gel electrophoresis using a 5-20% (w/v) gradientpolyacrylamide gel and detected on the gel as a single protein band,i.e., a high purity preparation.

Experiment 10 Properties of α-Glucosyltransferase from Arthrobacterglobiformis PP394 Experiment 10-1 Molecular Weight

The purified enzyme preparation of the α-glucosyltransferase fromArthrobacter globiformis PP349, obtained by the method in Experiment 9,was subjected to SDS-PAGE (a 5 to 20% (w/v) gradient gel) and themolecular weight of the enzyme was measured comparing with molecularweight markers, commercialized by Bio-Rad Japan, Tokyo, Japan. It wasrevealed that the α-glucosyltransferase has a molecular weight of90,000±10,000 daltons.

Experiment 10-2 Optimum Temperature and Optimum pH for the EnzymeReaction

Effects of temperature and pH on the enzyme activity were investigatedusing the purified enzyme preparation of the α-glucosyltransferase fromArthrobacter globiformis PP349, obtained by the method in Experiment 9,by varying temperature and pH at the assay of the enzyme. The resultsare in FIG. 7 (Optimum temperature) and FIG. 8 (Optimum pH),respectively. It was revealed that the optimum temperature of theα-glucosyltransferase was about 50° C. when reacted at pH 6.0 for 30 minand the optimum pH was about 6.0 when reacted at 40° C. for 30 min.

Experiment 7-3 Thermal and pH Stabilities of the Enzyme

Thermal stability and pH stability of the enzyme were investigated usingthe purified enzyme preparation of the α-glucosyltransferase, obtainedby the method in Experiment 9. Thermal stability of the enzyme wasdetermined by the steps of incubating an enzyme solution (20 mM acetatebuffer, pH 6.0) under various temperatures for 60 min, cooling in water,and measuring the residual enzyme activity. pH Stability of the enzymewas determined by the steps of incubating enzyme solution in 20 mMbuffer at various pHs, and at 4° C. for 24 hours, adjusting the pH to6.0, and measuring the residual enzyme activity. The results are in FIG.9 (Thermal stability) and in FIG. 10 (pH Stability), respectively. As isevident from the results in FIGS. 9 and 10, the α-glucosyltransferasefrom Arthrobacter globiformis of the present invention is stable up to40° C. and in the range of pH 4.0 to 8.0.

Experiment 7-4 Effects of Metal Ions on the Enzyme Activity

Effects of metal ions on the enzyme activity were investigated using thepurified enzyme preparation of the α-glucosyltransferase, obtained bythe method in Experiment 9, in the presence of 1 mM of respective metalions according to the assay method. The results are in Table 8.

TABLE 8 Metal Relative Metal Relative salt activity (%) salt activity(%) None 100 MgCl₂ 104 CaCl₂ 104 MnCl₂ 103 CoCl₂ 98 NiCl₂ 100 CuCl₂ 45ZnCl₂ 100 FeCl₂ 91 PbCl₂ 96 FeCl₃ 95 EDTA 99 HgCl₂ 2

As is evident from the results in Table 6, it was revealed that theα-glucosyltransferase activity was remarkably inhibited by Hg²⁺ ion andmoderately by Cu²⁺ ion, respectively.

Experiment 11 Action on Various Saccharides

Substrate specificity of the α-glucosyltransferase of the presentinvention was investigated using various saccharides as substrates.Substrate solutions were prepared by dissolving methyl-α-glucoside,methyl-β-glucoside, p-nitrophenyl-α-glucoside,p-nitrophenyl-β-glucoside, glucose, sucrose, maltose, isomaltose,trehalose, kojibiose, nigerose, neotrehalose, cellobiose, lactose,maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose,isomaltotriose, or isopanose, in water. Each substrate solution wasadmixed with acetate buffer (pH 6.0) to give a final concentration of 20mM, and then each of the resulting substrate solution was furtheradmixed with 10 units/g-solid-substrate of the purified preparation ofα-glucosyltransferase from Bacillus circulans PP710, obtained by themethod in Experiment 6. Successively, substrate concentration was set to1% (w/v) and followed by the enzyme reaction at 40° C. and pH 6.0 for 24hours. To examine the saccharides in each mixture before and after thereaction, saccharides were separated by silica gel thin-layerchromatography (hereinafter, simply abbreviated as “TLC”) using“KIESELGEL™ 60”, a TLC alminum plate (10×20 cm) and a solvent(n-butanol/pyridine/water, volume ratio of 6:4:1) and two-timesascending method. The formed saccharides except for the substrate on theplate were detected by visualizing the spots by spraying 10%sulfate-methanol solution and heating. By the above TLC analyses, theenzymatic action and the degree of the reaction of the enzyme on eachsubstrate were confirmed. The results are in Table 9. While, thesubstrate specificity of the α-glucosyltransferase from Arthrobacterglobiformis PP349 was also investigated by the same method describedabove, and it was confirmed that the substrate specificity of the enzymewas similar with that of the enzyme from Bacillus circulans PP710.

TABLE 9 Substrate Action* Substrate Action* Methyl-α-glucoside −Cellobiose − Methyl-β-glucoside − Sucrose − PNP*-α-glucoside − Lactose −PNP*-β-glucoside − Maltotriose ++ Glucose − Maltotetraose ++ Trehalose −Maltopentaose ++ Neotrehalose + Maltohexaose ++ Kojibiose ±Maltoheptaose ++ Nigerose + Isomaltotriose + Maltose ++ Isopanose +Isomaltose + Note: The symbol, “−” means “Not acted”. The symbol, “±”means “Slightly acted”. The symbol, “+” means “Acted”. The symbol, “++”means “Well acted”. *p-nitrophenyl

As is evident from the results in Table 9, the α-glucosyltransferase ofthe present invention acted on maltose, maltotriose, maltotetraose,maltopentaose, maltohexaose, and maltoheptaose, and also acted onnigerose, isomaltose, neothrehalose, isomaltotriose, and isopanose amongthe saccharides tested. Further, the enzyme slightly acted on kojibiose.From these saccharides, the formation of glucosyl-transfer products wasdetected together with hydrolyzates. While, the α-glucosyltransferase ofthe present invention did not act on methyl-α-glucoside,methyl-β-glucoside, p-nitrophenyl-α-glucoside,p-nitrophenyl-β-glucoside, trehalose, cellobiose, sucrose, lactose, etc.From these results and the fact that the α-glucosyltransferase forms thebranched α-glucan from partial starch hydrolyzate, it was revealed thatthe enzyme widely acts on maltose and α-1,4 glucan having a glucosepolymerization degree of 3 or higher, and α-gluco-oligosaccharidesconstructed by glucose molecules.

Experiment 12 Action Mechanism

For investigating the action mechanism of the α-glucosyltransferase ofthe present invention, the structure of the saccharide which is formedby allowing the enzyme to act on a minimum substrate, maltose, wasinvestigated. Since the result obtained by using theα-glucosyltransferase from Arthrobacter globiformis PP349 was almostsame with the case of using the enzyme from Bacillus circulans PP710,this experiment shows the results of the case of using the enzyme fromBacillus circulans PP710.

Experiment 12-1 Product from Maltose by the Enzyme Reaction

Aqueous maltose solution and acetate buffer (pH 6.0) were mixed to givefinal concentrations of 1% (w/v) and 10 mM, respectively, to make into asubstrate solution. The substrate solution was admixed with 10units/g-solid substrate of α-glucosyltransferase, obtained by the methodin Experiment 6, and followed by the enzyme reaction at 40° C. and pH6.0. Aliquots were sampled from the reaction mixture with time and thereaction was stopped by keeping at 100° C. for 10 min. The saccharidecompositions of the samples were measured by HPLC and GC. HPLC and GCwere carried out under the conditions described in Experiment 4-2. Theresults are in Table 10.

TABLE 10 Reaction Saccharide Composition (%, w/w) Time DP1 DP2 DP3 (hr)Glucose Maltose Isomaltose Neotrehalose Maltotriose Panose IsopanoseIsomaltotriose DP4 DP5 DP6≦ 0 0.0 99.2 0.0 0.0 0.8 0.0 0.0 0.0 0.0 0.00.0 1 12.1 55.0 0.7 0.0 13.2 16.1 0.0 0.0 2.9 0.0 0.0 2 18.4 33.5 1.00.0 14.4 24.1 0.0 0.0 7.6 1.0 0.0 4 23.7 17.3 2.5 0.0 10.0 29.3 0.0 0.013.0 4.2 0.0 8 27.0 10.4 4.7 0.0 4.9 27.8 0.0 0.0 15.9 5.3 4.0 24 30.46.0 10.0 2.6 1.4 11.0 1.0 3.6 15.3 10.2 8.5 48 31.5 6.2 13.8 3.9 0.7 4.11.5 4.2 12.4 10.3 11.4 DP: Glucose polymerization degree

As is evident from the results in Table 10, at initial stage (after onehour) of the reaction, glucose, maltotriose, and panose were formed asmajor reaction products from the substrate, maltose, by the action ofthe α-glucosyltransferase of the present invention. Also,oligosacchardes with a glucose polymerization degree of 4 and 5 wereformed when the reaction time was elapsed 2 to 4 hours. Accompanyingwith the progress of the reaction, the content of maltotriose reached amaximum, 14.4%, at 2 hours and then decreased; and the content of panosereached a maximum, 29.3% at 4 hours and then decreased; and the contentof isomaltose was increased with the decrease of the contents ofmaltotriose and panose. Further, the contents of isomaltose andoligosaccharides with a glucose polymerization degree of 4 or higherwere increased until 48 hours.

From these results, it was revealed that the α-glucosyltransferase ofthe present invention acts on maltose and forms glucose, maltotriose,and panose by catalyzing both α-1,4 and α-1,6 glucosyl transfer atinitial stage of the reaction; and forms isomaltose, which is formed byα-1,6 glucosyl transfer to glucose, and isopanose and isomaltotriose,which are formed by α-1,4 and α-1,6 glucosyl transfer to isomaltoseaccompanied with the progress of the reaction. Since the identificationof many kinds of oligosaccharide with glucose polymerization degree of 4or higher is difficult in this experiment, the reaction mechanism of theenzyme was investigated in the following Experiment 12-2 usingmaltopentaose, whose glucose polymerization degree is higher thanmaltose, as substrate.

Experiment 12-2 Product from Maltopentaose by the Enzyme Reaction

A substrate solution was prepared by mixing an aqueous maltopentaosesolution and acetate buffer (pH 6.0) to give final concentrations of 1%(w/v) and 10 mM, respectively. Successively, the resulting substratesolution was admixed with 10 units/g-solid substrate of theα-glucosyltransferase, obtained by the method in Experiment 6, andfollowed the enzyme reaction at pH 6.0 and 40° C. Time course of thereaction was investigated as follows: Samples of the reaction mixturewere withdrawn at various time and kept at 100° C. for 10 min to stopthe reaction. Saccharide compositions of the reaction mixtures weredetermined by HPLC. HPLC analysis was carried out under the conditionsdescribed in Experiment 4-2. In addition, the saccharides in thereaction mixture were subjected to the conventional methylation analysisand the partially methylated products were analyzed bygas-chromatography, and the ratio of various glucosidic linkages in thesaccharide was determined based on the composition of the partiallymethylated products. Further, the saccharides in the reaction mixturewere subjected to the isomaltodextranase digestion similarly inExperiment 4-2. Time course of the saccharide composition in thereaction mixture is in Table 11. The results of the methylation analysisand the isomaltodextranase digestion are in Table 12.

TABLE 11 Reac- tion time Saccharide Composition (%, w/w) (hr) DP1 DP2DP3 DP4 DP5 DP6 DP7 DP8 DP9≦ 0 0.0 0.0 0.3 1.2 97.4 0.1 0.0 0.0 0.0 10.4 1.0 3.4 15.0 53.4 21.9 3.3 0.8 0.1 4 1.2 2.0 7.5 15.2 27.1 25.5 12.35.7 3.0 8 2.9 3.4 8.9 12.6 18.2 20.0 14.2 8.9 9.7 24 6.9 3.7 8.1 8.212.5 14.2 13.9 11.2 21.1 DP: Glucose polymerization degree

TABLE 12 Composition of partially methylated product (Peak area %)3,4,6-Tri- 2,4,6-Tri- 2,3,6-Tri- 2,3,4-Tri- 2,4-Di- 2,3-Di-2,3,4,6-Tetra- methylated methylated methylated methylated methylatedmethylated methylated product product product product product productReaction product Glc Glc Glc Glc Glc Glc Isomaltose Time Non-reducinginvolving involving involving involving involving involving content*(hr) end Glc** 1,2-linkage 1,3-linkage 1,4-linkage 1,6-linkage1,3,6-linkage 1,4,6-linkage (%, w/w) 0 20.0 0.0 0.0 80.0 0.0 0.0 0.0 0.01 20.9 0.0 0.1 76.9 1.7 0.0 0.4 2.7 4 24.2 0.0 0.8 67.5 7.2 0.0 0.3 11.98 26.1 0.0 1.0 55.4 16.1 0.7 0.8 23.3 24 28.6 0.0 0.8 37.0 30.8 1.6 1.240.9 *After isomaltodextranase digestion **Glucose residue

As is evident from the results in Tables 11 and 12, at initial stage(after one hour) of the reaction, an oligosaccharide whose glucosepolymerization degree is larger by one than that of the substrate andthat whose glucose polymerization degree is smaller by one than thesubstrate were formed preferentially. Based on the fact, it wasconfirmed that the enzyme catalyzes the transglucosylation. Accompanyingwith the progress of the reaction, reaction products with variousglucose polymerization degrees were formed, and the content of glucanswith a glucose polymerization degree of 9 or higher reached to 21.1% at24 hours. From the results of the methylation analysis, it was reveledthat accompanying with the progress of the reaction, the content ofglucose residue involving 1,4 linkage was decreased with the significantincrease of the content of glucose residue involving 1,6 linkage; andthe contents of glucose residue involving 1,3 linkage, that involving1,4,6 linkage, and that involving 1,3,6 linkage were gradually increasedin the reaction products. It was also revealed that the isomaltosecontent after the isomaltodextranase digestion of the reaction productwas significantly increased accompanying with the progress of thereaction. Almost the same results were obtained in the case of using theα-glucosyltransferase from Arthrobacter globiformis PP349 for the sametests.

From the results in Experiments 12-1 and 12-2, the reaction mechanism ofthe α-glucosyltransferase of the present invention was estimated asfollows:

(1) The enzyme acts on maltose and/or α-1,4 glucan having a glucosepolymerization degree of 3 or higher as the substrate and forms theα-1,4 glucan in which a glucose residue is bound via α-linkage tohydroxyl group at C-4 or C-6 position of the non-reducing end glucoseresidue (α-glucan whose glucose polymerization degree is increased byone) and the α-1,4 glucan whose glucose polymerization degree isdecreased by one, by mainly transferring the non-reducing end glucoseresidue to the non-reducing end glucose residue of the other α-1,4glucan by α-1,4 or α-1,6 transglucosylation.(2) The enzyme further acts on the α-1,4 glucan whose glucosepolymerization degree is decreased by one, formed in the above step (1);and transfers a glucose residue to the C-4 or C-6 hydroxyl group of thenon-reducing end glucose residue of the α-glucan whose glucosepolymerization degree is increased by one, also formed in the above step(1), by the intermolecular α-1,4 or α-1,6 transglucosylation to elongatethe glucose chain.(3) By repeating the reactions in the above steps (1) and (2), theenzyme forms a glucan having both α-1,4 and α-1,6 linkages from maltoseand/or α-glucan having a glucose polymerization degree of 3 or higher.(4) Although the frequency is low, the enzyme forms a glucan havingα-1,3, α-1,4,6, and α-1,3,6 linkages in addition to α-1,4 and α-1,6linkages by catalyzing the α-1,3 transglucosylation and α-1,4 or α-1,3transglucosylation to the internal glucose residues involving α-1,6linkages, in the glucan.(5) As results of repeating the reactions in the above steps (1) to (4),the branched α-glucan of the present invention, in which glucose ismainly bound via α-1,4 and α-1,6 linkages and which has α-1,3, α-1,4,6,and α-1,3,6 linkages in a low frequency, is formed by the enzyme.

Experiment 13 α-Glucosyl-Transferring Reaction and the Change ofReducing Power of the Reaction Mixture

Substrate solutions were prepared by mixing an aqueous maltose solutionand acetate buffer (pH 6.0) to give final concentrations of 1% or 30%(w/v) and 20 mM, respectively. Successively, the resulting substratesolution was admixed with 4 units/g-solid substrate of the purifiedpreparation of the α-glucosyltransferase from Bacillus circulans PP710,obtained by the method in Experiment 6, and followed the enzyme reactionat pH 6.0 and 40° C. Time course of the reaction was investigated asfollows: Samples of the reaction mixture were withdrawn at various timeand kept at 100° C. for 10 min to stop the reaction. The amount ofresidual maltose in the reaction mixtures were determined by HPLC and GCdescribed in Experiment 4-2. The amounts of reducing saccharide andtotal saccharide were determined by Somogyi-Nelson method andAnthrone-sulfuric acid method, respectively; and then the reducing powerof the reaction mixture was calculated using the following formula:

Reducing power={(The amount of reducing saccharide/The amount of totalsaccharide)}×100

The results are in Table 13.

TABLE 13 Concentration of maltose 1% (w/w) 30% (w/w) Amount of Amount ofReaction residual Reducing residual Reducing time maltose power maltosepower (hr) (%) (Relative %) (%) (Relative %) 0 99.7 43.6 99.7 43.6(100%) (100%) 4 64.2 45.0 68.8 43.6 (103%) (100%) 8 43.0 46.0 51.8 43.6(106%) (100%) 16 20.9 46.7 28.8 43.7 (107%) (100%) 24 8.1 47.9 13.3 43.8(109%) (101%)

As is evident from the results in Table 13, when theα-glucosyltransferase of the present invention was allowed to act onmaltose, reducing power of the reaction mixture was slightly increasedin the case of using the substrate with a relatively low concentration,1% (w/v), and substantially not increased in the case of using thesubstrate with a relatively high concentration, 30% (w/v). The fact thatthe reducing power of the reaction mixture was only slightly increasedin the case of the relatively low substrate concentration and the amountof residual maltose was less than 10%, means that theα-glucosyltransferase of the present invention is an enzyme inherentlycatalyzing the transferring reaction and hardly hydrolyzes the substrateduring the reaction. Almost the same results were obtained in the caseof using the α-glucosyltransferase from Arthrobacter globiformis PP349for the same tests.

Experiment 14 Comparison of the Purified α-Glucosyltransferase and CrudeEnzyme in the Formation of the Branched α-Glucan

From the viewpoint of industrial production of the branched α-glucan,the crude enzyme of the α-glucosyltransferase is preferable as far as itcan be used for the production because it is not necessary to purify theenzyme. Accordingly, it was investigated whether the branched α-glucanhaving almost equal characteristics with Glucan A, prepared inExperiment 1-2, can be obtained or not by using the crude enzyme of theα-glucosyltransferase from Bacillus circulans. The crudeα-glucosyltransferase solution was prepared by the steps of culturingBacillus circulans PP710 by the method in Experiment 1-1, salting outthe resulting culture supernatant using ammonium sulfate, and dialyzingthe resulting precipitates against 20 mM acetate buffer (pH 4.5). Thecrude enzyme solution was allowed to act on “PINEDEX #100®”, a partialstarch hydrolyzate commercialized by Matsutani Chemical Industries Co.,Ltd., Hyogo, Japan, and a glucan solution with a concentration of 30%(w/w) was obtained from the partial starch hydrolyzate as substrate in ayield of 88.2%, on a dry solid basis. The resulting branched α-glucanwas called as “Glucan C” and subjected to the methylation analysis. Theresult is in Table 14. The molecular weight distribution and the WSDFcontent of Glucan C, measured by the methods described in Experiments 3and 4, are in Table 15. In Tables 14 and 15, the data of the partialstarch hydrolyzate used as material and Glucan A prepared by usingpartially purified α-glucosyltransferase were copied from Tables 3 and 4as comparative data.

TABLE 14 Composition (Peak area %) Partial starch hydrolyzate* Partiallymethylated product Corresponding Glc** (Material) Glucan A Glucan C2,3,4,6-Tetramethylated product Non-reducing end Glc 5.8 10.1 16.03,4,6-Trimethylated product Glc involving 1,2-linkage 0.0 0.0 0.02,4,6-Trimethylated product Glc involving 1,3-linkage 0.0 1.1 3.02,3,6-Trimethylated product Glc involving 1,4-linkage 89.8 51.5 30.02,3,4-Trimethylated product Glc involving 1,6-linkage 0.0 32.2 40.32,4-Dimethylated product Glc involving 1,3,6-linkage 0.0 0.8 4.82,3-Dimethylated product Glc involving 1,4,6-linkage 4.4 4.5 5.8*“PINEDEX ® #100”, a partial starch hydrolyzate commercialized byMatsutani Chemical Industries Co., Ltd., Hyogo, Japan. **Glucose residueGlucan A: Glucan prepared by using α-glucosyltransferase (partiallypurified enzyme) from strain PP710 Glucan C: Glucan prepared by usingα-glucosyltransferase (crude enzyme) from strain PP7

TABLE 15 Partial starch hydrolyzate* Analytical data (Material) Glucan AGlucan C Number average molecular 6,680 3,840 2,840 weight (Mn) (Dalton)Weight-average molecular 98,890 59,000 6,220 weight (Mw) (Dalton) Mw/Mn14.8 15.4 2.2 Average glucose polymerization 449 and 6.3 384, 22.2, 26.7and 1 degree of peaks 10.9, and 1 WSDF content (%) 0.0 42.1 75.8*“PINEDEX ® #100”, a partial starch hydrolyzate commercialized byMatsutani Chemical Industries Co., Ltd., Hyogo, Japan. Glucan A: Glucanprepared by using α-glucosyltransferase (partially purified enzyme) fromstrain PP710 Glucan C: Glucan prepared by using α-glucosyltransferase(crude enzyme) from strain PP710

As shown in Table 14, in the case of Glucan C, prepared by using thecrude enzyme preparation of the α-glucosyltransferase from Bacilluscirculans PP710, the contents of 2,3,4,6-tetramethylated product,corresponding to non-reducing end glucose residue, and2,3,4-trimethylated product, corresponding to glucose residue involvingto α-1,6 linkage, were unexpectedly increased in the methylationanalysis in comparison with the case of Glucan A. The results indicatethat Glucan C has many non-reducing ends and contains much more α-1,6linkages than Glucan A. Further, in Glucan C, the content of2,4-dimethylated product was 4.8%, which is larger than that of GlucanA, 0.8%. The result indicates that glucose residue involving both 1,3linkage and 1,6 linkage is increased in Glucan C.

As is evident from Table 15, Glucan C showed lower values of numberaverage molecular weight and weight-average molecular weight (lowerglucose polymerization degree), and significantly larger WSDF contentthan Glucan A. These results suggest that another enzyme different fromthe α-glucosyltransferase is concomitant in the crude enzyme preparationof the α-glucosyltransferase from Bacillus circulans PP710, and theconcomitant enzyme involves the increase of α-1,6 linkage and glucoseresidue involving both 1,3 linkage and 1,6 linkage; the lowering themolecular weight; and the increase of the WSDF content.

Experiment 15 Characterization and Purification of the ConcomitantEnzyme in the Crude Enzyme Preparation of α-Glucosyltransferase fromBacillus circulans PP710

The enzyme, which co-exists in the crude enzyme preparation ofα-glucosyltransferase from Bacillus circulans PP710 and involves theincrease of α-1,6 linkages and glucose residue involving both 1,3 and1,6 linkages, the lowering of the molecular weight, and the increase ofthe content of WSDF; was characterized and purified in the followingexperiments.

Experiment 15-1 Characterization of the Concomitant Enzyme and Assay ofthe Enzyme Activity

Bacillus circulans PP710, FERM BP-10771, was cultivated by the method inExperiment 1-1, and about 3 L of the resulting culture supernatant wassalted out using ammonium sulfate. Then, the resulting precipitated wasdissolved in water, dialyzed against 20 mM Tris-HCl buffer containing 1mM CaCl₂, and about 40 ml of the resulting dialyzate was collected as acrude enzyme solution. The crude enzyme solution was admixed with 2%(w/v) of soluble starch solution or 2% (w/v) of pullulan solution, andfollowed by the enzyme reaction at pH 6.0 and 40° C. for 16 hours. Afterstopping the reaction by heating at 100° C. for 10 min, the reactionmixture was subjected to TLC using “SILICAGEL™ 60F₂₅₄”, a TLC plate(10×20 cm) commercialized by Merck. TLC was carried out using a solvent(n-butanol/pyridine/water, volume ratio of 6:4:1) and two-timesascending method. The products on the plate were detected by visualizingthe spots by spraying 20% sulfate-methanol solution and heating at 100°C. for 5 min. By the TLC analyses, it was revealed that maltose andα-1,4 glucan having a glucose polymerization degree of 3 or higher wereformed from soluble starch, and slight amounts of isomaltose and panosewere formed from pullulan. It was revealed that an amylase whichhydrolyzes starch and pullulan was present in the crude enzyme fromBacillus circulans PP710, FERM BP-10771, as a concomitant enzyme.

The activity of the concomitant amylase was assayed as follows: Asubstrate solution is prepared by dissolving “AMYLOSE EX-I”, a shortchain amylose with average glucose polymerization degree of 17,commercialized by Hayashibara Biochemical Laboratories Inc., Okayama,Japan, into 50 mM acetate buffer (pH 6.0) containing 1 mM CaCl₂ to givea final concentration of 1% (w/v). To 2 ml of the substrate solution,0.2 ml of the enzyme solution is admixed and followed by the enzymereaction at 35° C. for 30 min. After the reaction, 0.2 ml of thereaction mixture is withdrawn and mixed with 8 ml of 0.02 N sulfuricacid aqueous solution to stop the reaction. After adding 0.2 ml of 0.1 Niodine solution to the resulting solution, the resulting mixture is keptat 25° C. for 15 min and the absorbance at 660 nm of the mixture ismeasured. Separately, a reaction mixture at zero time-reaction istreated by the same manner and the absorbance at 660 nm of the mixtureis measured. By using the both values, the decrease of iodine-stain perminute is calculated. One unit of the amylase activity is defined as the10-fold amount of enzyme which decreases 10% of the absorbance at 660 nm(iodine-stain), corresponding to 20 mg of short chain amylose, under theabove conditions.

Experiment 15-2 Purification of the Concomitant Amylase

The crude enzyme solution, prepared in Experiment 15-1, was subjected toanion-exchange column chromatography using 70 ml of “DEAF-TOYOPEARL™650S” gel, a gel commercialized by Tosoh Corporation, Tokyo, Japan. Theamylase activity was not adsorbed on “DEAF-TOYOPEARL™ 650S” gelpre-equilibrated with 20 mM Tris-HCl buffer (pH 7.5) containing 1 mMCaCl₂ and eluted as non-absorbing fractions. The active fractions werecollected, added ammonium sulfate to give a final concentration of 1.5M,and stand at 4° C. for 24 hours. Successively, the enzyme solution wascentrifuged to remove insoluble substances and subjected to hydrophobiccolumn chromatography using 1 ml of “RESOURCE™ PHE” gel, a gelcommercialized by Pharmacia Biotech. The amylase activity was adsorbedon “RESOURCE™ PHE” gel pre-equilibrated with 20 mM Tris-HCl buffer (pH7.5) containing 1.5 M ammonium sulfate and 1 mM CaCl₂ and when elutedwith a linear gradient decreasing from 1.5 M to zero M of ammoniumsulfate, the enzyme activity was eluted at about 0.3 M of ammoniumsulfate. The active fractions were collected, concentrated, andsubjected to gel-filtration column chromatography using 118 ml of“SUPERDEX™ 200pg” gel, a gel commercialized by Pharmacia Biotech. Theamylase activity was eluted using 20 mM Tris-HCl buffer (pH 7.5)containing 0.2 M sodium chloride and 1 mM CaCl₂. The active fractionswere collected and subjected to anion-exchange column chromatographyusing 1 ml of “RESOURCE™ Q” gel, a gel commercialized by PharmaciaBiotech. The amylase activity was not adsorbed on “RESOURCE™ Q” gelpre-equilibrated with 20 mM Tris-HCl buffer (pH 7.5) containing 1 mMCaCl₂ and eluted as non-absorbing fractions. The active fractions werecollected as the purified amylase preparation. The amount of enzymeactivity, specific activity, and yield of the amylase at eachpurification step are in Table 16.

TABLE 16 Amylase Specific activity activity of amylase YieldPurification step (units) (units/mg-protein) (%) Dialyzed solution after358 3.4 100 salting out with ammonium sulfate Eluate from ion-exchange174 30.9 48.6 column chromatography Eluate from hydrophobic 56.8 42.515.9 column chromatography Eluate from gel-filtration 34.4 51.8 9.6column chromatography Eluate from ion-exchange 32.8 52.6 9.2 columnchromatography

The finally purified enzyme preparation of the amylase was assayed forpurity on gel electrophoresis using a 5-20% (w/v) gradientpolyacrylamide gel and detected on the gel as a single protein band,i.e., a high purity preparation.

Experiment 16 Properties of the Amylase from Bacillus circulans PP710Experiment 16-1 Molecular Weight

The purified enzyme preparation of the amylase, obtained by the methodin Experiment 15-2, was subjected to SDS-PAGE (a 5 to 20% (w/v) gradientgel) and the molecular weight of the enzyme was measured comparing withmolecular weight markers, commercialized by Bio-Rad Japan, Tokyo, Japan.It was revealed that the amylase has a molecular weight of 58,000±10,000daltons.

Experiment 16-2 Optimum Temperature and pH of the Amylase

Effects of temperature and pH on the enzyme activity were investigatedusing the purified enzyme preparation of the amylase from Bacilluscirculans PP710, obtained by the method in Experiment 15-2, by varyingtemperature and pH at the assay of the enzyme. The results are in FIG.11 (Optimum temperature) and FIG. 12 (Optimum pH), respectively. It wasrevealed that the optimum temperature of the amylase was about 55° C.when reacted at pH 6.0 for 30 min and the optimum pH was 6.0 to 7.0 whenreacted at 35° C. for 30 min.

Experiment 16-3 Thermal and pH Stabilities of the Amylase

Thermal stability and pH stability of the enzyme were investigated usingthe purified enzyme preparation of the amylase, obtained by the methodin Experiment 15-2. Thermal stability of the enzyme was determined bythe steps of incubating an enzyme solution (20 mM acetate buffer, pH6.0, or the same buffer containing 1 mM CaCl₂) under varioustemperatures for 60 min, cooling in water, and measuring the residualenzyme activity. pH Stability of the enzyme was determined by the stepsof incubating enzyme solution in 20 mM buffer at various pHs, and at 4°C. for 24 hours, adjusting the pH to 6.0, and measuring the residualenzyme activity. The results are in FIG. 13 (Thermal stability) and inFIG. 14 (pH Stability), respectively. As is evident from the results inFIGS. 13 and 14, the amylase is stable up to 40° C. in the absence ofcalcium ion and up to 50° C. in the presence of 1 mM calcium ion, andstable in the range of pH 6.0 to 8.0.

Experiment 16-5 Substrate Specificity of the Amylase

The action of the amylase to various substrates was investigated usingthe purified preparation of the amylase, obtained by the method inExperiment 15-2. As a result, it was revealed that the amylase hydrolyzestarch, maltose, and α-1,4 glucan having a glucose polymerization degreeof 3 or higher, and also catalyzes the transglycosylation. It was alsorevealed that the amylase forms cyclodextrins from starch and formspanose by hydrolyzing pullulan.

Experiment 17 Preparation of the Branched α-Glucan by Usingα-Glucosyltransferase and the Amylase in Combination

Using the purified preparation of the α-glucosyltransferase fromBacillus circulans PP710, obtained by the method in Experiment 6, andthe purified preparation of the amylase, obtained by the method inExperiment 15-2, it was investigated whether Glucan C in Experiment 14,which was produced by using the crude enzyme preparation ofα-glucosyltransferase from Bacillus circulans PP710, can be re-producedor not. “PINEDEX® #100”, a partial starch hydrolyzate commercialized byMatsutani Chemical Industries Co., Ltd., Hyogo, Japan, was dissolved inwater to give a concentration of 30% (w/w) and the pH of the solutionwas adjusted to 6.0. To the substrate solution, 10 units/g-solid of thepurified preparation of the α-glucosyltransferase from Bacilluscriculans PP710, obtained by the method in Experiment 6; and zero, 0.1,0.2, 0.5 or 1.0 unit/g-solid of the amylase obtained by the method in15-2 were added, and followed by the enzyme reaction at pH 6.0 and 40°C. for 72 hours. After completion of the reaction, the reaction mixturewas boiled for 10 min to stop the reaction. The reaction mixturescontaining the branched α-glucan, obtained by respective reactioncondition, were subjected to gel-filtration HPLC described in Experiment4-4, and the chromatograms are shown in FIG. 15 together with that ofthe partial starch hydrolyzate used as a substrate. In FIG. 15, symbol“a” is the gel-filtration HPLC chromatogram of partial starchhydrolyzate used as a substrate, and symbols “b”, “c”, “d”, and “e” arethose of the branched α-glucans obtained by using 10 units/g-solid ofthe α-glucosyltransferase and 0.1, 0.2, 0.5, or 1.0 unit/g-solid of theamylase, respectively. (Since the gel-filtration HPLC chromatogram ofthe branched α-glucan, obtained by using 10 units/g-solid of theα-glucosyltransferase only, is almost same with that of Glucan A in FIG.1, it is omitted in FIG. 15, and also in the following FIGS. 16 to 19.)The results of the molecular weight distribution analyses based on thosegel-filtration HPLC chromatograms and the WSDF content measured by theEnzyme-HPLC method in Experiment 3 of the branched α-glucans are inTable 17. Those of the partial starch hydrolyzate used as a substrate(zero unit/g-solid of α-glucosyltransferase, and zero unit of amylase)are also in Table 17.

TABLE 17 Molecular weight distribution of glucan Number Weight- averageaverage Amount of enzyme molecular molecular (unit/g-substrate) weightweight WSDF α-Glucosyl (Mn) (Mw) content tansferase Amylase (Dalton)(Dalton) Mw/Mn (%, w/w) 0 0.0 6,670 100,340 15.0 0.0 10 0.0 5,530 97,45017.6 41.1 10 0.1 3,235 7,799 2.4 71.5 10 0.2 3,013 6,627 2.2 73.7 10 0.52,789 5,894 2.1 76.7 10 1.0 2,544 5,304 2.1 76.8

As is evident from the results in Table 17, in the case of using theα-glucosyltransferase and the amylase in combination, the molecularweight of the formed branched α-glucan was decreased and the WSDFcontent was significantly increased. The number average molecularweight, the weight-average molecular weight, and the value of dividingthe weight-average molecular weight with the number average molecularweight, Mw/Mn, of the branched α-glucan were decreased with increase ofthe amount of the amylase. From the results, it was revealed that therange of the molecular weight distribution of the branched α-glucanbecame narrower with increase of the amount of the amylase. In the caseof using 0.5 unit/g-solid of the amylase, the value of Mw/Mn wasdecreased to 2.1. Further, in the cases of using 0.5 unit/g-solid orhigher of the amylase, the WSDF contents of the branched α-glucan werereached to about 76% (w/w).

From the results, it was confirmed that the decrease of the molecularweight and the increase of the WSDF content in Glucan C, obtained inExperiment 14 by using the crude enzyme preparation of theα-glucosyltransferase from Bacillus circulans PP710, were caused by theconcomitant amylase in the crude enzyme preparation. It is consideredthat the concomitant amylase partially hydrolyzed the partial starchhydrolyzate as a substrate and the branched α-glucan as a product by theα-glucosyltransferase to decrease the molecular weight; and acts forincreasing the WSDF content by transferring glycosyl group to thebranched α-glucan. Further, the results indicate that the branchedα-glucan, having a low molecular weight and a high WSDF content, can beproduced by using the amylase and the α-glucosyltransferase of thepresent invention in combination.

Experiment 18 Preparations of the Branched α-Glucan by Using theα-Glucosyltransferase and Other Well-Known Amylases in Combination

Various branched α-glucans were prepared by allowing theα-glucosyltransferase of the present invention and other well-knownamylases to act in combination on the partial starch hydrolyzate, andthe characteristics and the WSDF contents of the resulting branchedα-glucan were investigated. Almost equal results were obtained in thecases of using the α-glucosyltransferase from Bacillus circulans PP710and from Arthrobacter globiformis PP349. Therefore, in this experiment,the results obtained by using the α-glucosyltransferase from Bacilluscirculans PP710 were described.

Experiment 18-1 Preparation of the Branched α-Glucan by Usingα-Glucosyltransferase and Isoamylase in Combination; and MolecularWeight Distribution and the WSDF Content of the Resulting Branchedα-Glucan

Except for using zero, 50,200, 500, or 1,000 units/g-solid of isoamylasefrom Pseudomonas amyloderamosa, prepared by Hayashibara BiochemicalLaboratories Inc., Okayama, Japan, as a substitute of the amylase fromBacillus circulans PP710, the branched α-glucan was prepared accordingto the method in Experiment 17. Each branched α-glucan, prepared by eachreaction condition, was subjected to the gel-filtration HPLC inExperiment 4-4, and the chromatogram was shown in FIG. 16 together withthat of the partial starch hydrolyzate used as substrate. In FIG. 16,symbol “a” is a gel-filtration HPLC chromatogram of the partial starchhydrolyzate used as substrate, and symbols “b”, “c”, “d”, and “e” aregel-filtration HPLC chromatograms of the branched α-glucans obtained byusing 10 units/g-solid of the α-glucosyltransferase, and 50, 200, 500,and 1,000 units/g-solid of isoamylase in combination, respectively. Theresults of the molecular weight distribution analyses based on thegel-filtration HPLC chromatograms and the WSDF contents determined byEnzyme-HPLC method in Experiment 3 are in Table 18.

TABLE 18 Molecular weight distribution of glucan Number Weight- Amountof enzyme average average (unit/g-substrate) molecular molecular α-weight weight WSDF Glucosyl (Mn) (Mw) content tansferase Isoamylase(Dalton) (Dalton) Mw/Mn (%, w/w) 0 0 6,670 100,340 15.0 0.0 10 0 5,53097,450 17.6 41.1 10 50 3,490 19,980 5.7 40.6 10 200 2,560 6,580 2.6 42.710 500 2,230 4,340 1.9 43.7 10 1,000 2,140 4,020 1.9 41.6

As is evident from the result in the case of allowing theα-glucosyltransferase only to act on the partial starch hydrolyzate inTable 18, the α-glucosyltransferase did not exercise an influence on themolecular weight distribution of the branched α-glucan. However, as isevident from the results in Table 18 and FIG. 16, both the numberaverage molecular weight and the weight-average molecular weight, andMw/Mn, the value of dividing the weight-average molecular weight withthe number average molecular weight were decreased with increase of theamount of isoamylase. From the results, it was revealed that the rangeof the molecular weight distribution of the branched α-glucan becamenarrower by the action of isoamylase. In the case of using 1,000units/g-solid of isoamylase, Mw/Mn of the branched α-glucan wasdecreased to 1.9 and the branched α-glucan showed a molecular weightdistribution with a peak at glucose polymerization degree of 20.2.While, the WSDF contents of the branched α-glucans, obtained by thereaction conditions, were not influenced by the amount of isoamylase andwere about 40 to 44% (w/w).

From these results, it was revealed that the branched α-glucan with alow molecular weight can be produced without altering the WSDF contentby allowing the α-glucosyltransferase of the present invention andisoamylase to act in combination on the partial starch hydrolyzate.

Experiment 18-2 Preparation of the Branched α-Glucan by Usingα-Glucosyltransferase and α-Amylase in Combination; and Molecular WeightDistribution and the WSDF Content of the Resulting Branched α-Glucan

Except for using zero, 0.1, 0.2, 0.5, or 1.0 unit/g-solid of “NEOSPITASEPK2”, an α-amylase commercialized by Nagase ChemteX Corporation, Osaka,Japan, as a substitute of isoamylase from Pseudomonas amyloderamosa, thebranched α-glucan was prepared according to the method in Experiment18-1. Each branched α-glucan, prepared by each reaction condition, wassubjected to the gel-filtration HPLC in Experiment 4-4, and thechromatogram was shown in FIG. 17 together with that of the partialstarch hydrolyzate used as substrate. In FIG. 17, symbol “a” is agel-filtration HPLC chromatogram of the partial starch hydrolyzate usedas substrate, and symbols “b”, “c”, “d”, and “e” are gel-filtration HPLCchromatograms of the branched α-glucans obtained by using 10units/g-solid of the α-glucosyltransferase, and 0.1, 0.2, 0.5, and 1.0unit/g-solid of α-amylase in combination, respectively. The results ofthe molecular weight distribution analyses based on the gel-filtrationHPLC chromatograms and the WSDF contents determined by Enzyme-HPLCmethod in Experiment 3 are in Table 19.

TABLE 19 Molecular weight distribution of glucan Number Weight- Amountof enzyme average average (unit/g-substrate) molecular molecular α-weight weight WSDF Glucosyl (Mn) (Mw) content tansferase α-Amylase(Dalton) (Dalton) Mw/Mn (%, w/w) 0 0.0 6,670 100,340 15.0 0.0 10 0.05,530 97,450 17.6 41.1 10 0.1 2,835 15,698 5.5 48.4 10 0.2 2,189 8,6564.0 51.7 10 0.5 1,694 4,755 2.8 53.5 10 1.0 1,475 3,552 2.4 54.0

As is evident from the results in FIG. 17 and Table 19, both the numberaverage molecular weight and the weight-average molecular weight, andMw/Mn, the value of dividing the weight-average molecular weight withthe number average molecular weight were decreased with increase of theamount of α-amylase. From the results, it was revealed that the range ofthe molecular weight distribution of the branched α-glucan becamenarrower by the action of α-amylase. In the case of using 1.0unit/g-solid of α-amylase (Symbol “e” in FIG. 17), Mw/Mn of the branchedα-glucan was decreased to 2.4 and the branched α-glucan showed amolecular weight distribution with a peak at glucose polymerizationdegree of 11.8. While, the WSDF contents of the branched α-glucans,obtained by the reaction conditions, showed a tendency of increasingwith increase of the amount of α-amylase.

From these results, it was revealed that the branched α-glucan with alow molecular weight and increased WSDF content can be produced byallowing the α-glucosyltransferase of the present invention andα-amylase to act in combination on the partial starch hydrolyzate.

Experiment 18-3 Preparation of the Branched α-Glucan by Usingα-Glucosyltransferase and CGTase in Combination; and Molecular WeightDistribution and the WSDF Content of the Resulting Branched α-Glucan

Except for using zero, 0.1, 0.2, 0.5, or 1.0 unit/g-solid of CGTase fromBacillus stearothermophilus, produced by Hayashibara BiochemicalLaboratories Inc., Okayama, Japan, as a substitute of isoamylase fromPseudomonas amyloderamosa, the branched α-glucan was prepared accordingto the method in Experiment 18-1. Each branched α-glucan, prepared byeach reaction condition, was subjected to the gel-filtration HPLC inExperiment 4-4, and the chromatogram was shown in FIG. 18 together withthat of the partial starch hydrolyzate used as substrate. In FIG. 18,symbol “a” is a gel-filtration HPLC chromatogram of the partial starchhydrolyzate used as substrate, and symbols “b”, “c”, “d”, and “e” aregel-filtration HPLC chromatograms of the branched α-glucans obtained byusing 10 units/g-solid of the α-glucosyltransferase, and 0.1, 0.2, 0.5,and 1.0 unit/g-solid of CGTase in combination, respectively. The resultsof the molecular weight distribution analyses based on thegel-filtration HPLC chromatograms and the WSDF contents determined byEnzyme-HPLC method in Experiment 3 are in Table 20.

TABLE 20 Molecular weight distribution of glucan Number Weight- averageaverage Amount of enzyme molecular molecular (unit/g-substrate) weightweight WSDF α-Glucosyl (Mn) (Mw) content tansferase CGTase (Dalton)(Dalton) Mw/Mn (%, w/w) 0 0.0 6,670 100,340 15.0 0.0 10 0.0 5,530 97,45017.6 39.1 10 0.1 4,733 32,833 6.9 57.5 10 0.2 4,733 23,418 4.9 62.8 100.5 4,501 16,357 3.6 68.2 10 1.0 4,401 14,107 3.2 70.5 CGTase:Cyclomaltodextrin glucanotransferase

As is evident from the results in FIG. 18 and Table 20, both the numberaverage molecular weight and the weight-average molecular weight, andMw/Mn, the value of dividing the weight-average molecular weight withthe number average molecular weight were decreased with increase of theamount of CGTase. From the results, it was revealed that the range ofthe molecular weight distribution of the branched α-glucan becamenarrower by the action of CGTase. In the case of using 1.0 unit/g-solidof CGTase (Symbol “e” in FIG. 18), Mw/Mn of the branched α-glucan wasdecreased to 3.2 and the branched α-glucan showed a molecular weightdistribution with a peak at glucose polymerization degree of 79.1.While, the WSDF contents of the branched α-glucans, obtained by thereaction conditions, showed a tendency of increasing with increase ofthe amount of CGTase. The WSDF content of the branched α-glucan,obtained by using CGTase in combination, was significantly increased incomparison with the case of using α-amylase in Experiment 18-2. In thecase of using 1.0 unit/g-solid of CGTase, the WSDF content of thebranched α-glucan was reached to 70.5% (w/w).

From these results, it was revealed that the branched α-glucan with alow molecular weight and significantly increased WSDF content can beproduced by allowing the α-glucosyltransferase of the present inventionand CGTase to act in combination on the partial starch hydrolyzate.Since CGTase is an enzyme catalyzing the hydrolysis of α-1,4 linkage andalso the glycosyl-transfer, it forms many non-reducing end glucoseresidues in the branched α-glucan without significantly lowering themolecular weight in comparison with the case of using α-amylase.Therefore, it is suggested that the α-glucosyltransferase is able to actmore frequently on the branched α-glucan formed by using CGTase thanthat formed by using α-amylase, and as a result, the branched α-glucanwith increased WSDF content can be obtained.

Experiment 18-4 Preparation of the Branched α-Glucan by Usingα-Glucosyltransferase, Isomalyase, and CGTase in Combination; andMolecular Weight Distribution and the WSDF Content of the ResultingBranched α-Glucan

Except for further adding zero or 1.0 unit/g-solid of CGTase fromBacillus stearothermophilus to the reaction system in Experiment 18-1,the branched α-glucan was prepared according to the method in Experiment18-1. Each branched α-glucan, prepared by each reaction condition, wassubjected to the gel-filtration HPLC in Experiment 4-4, and thechromatogram was shown in FIG. 19 together with that of the partialstarch hydrolyzate used as substrate. In FIG. 19, symbol “a” is agel-filtration HPLC chromatogram of the partial starch hydrolyzate usedas substrate, and symbols “b”, “c”, “d”, and “e” are gel-filtration HPLCchromatograms of the branched α-glucans obtained by using 10units/g-solid and 1.0 unit/g-solid of the α-glucosyltransferase andCGTase, respectively, and 50, 200, 500, and 1,000 units/g-solid ofisoamylase in combination, respectively. The results of the molecularweight distribution analyses based on the gel-filtration HPLCchromatograms and the WSDF contents determined by Enzyme-HPLC method inExperiment 3 are in Table 21.

TABLE 21 Molecular weight distribution of glucan Amount of enzyme(unit/g-substrate) Number average Weight-average WSDF α-Glucosylmolecular weight molecular weight content tansferase Isoamylase CGTase(Mn) (Dalton) (Mw) (Dalton) Mw/Mn (%, w/w) 0 0 0.0 6,670 100,340 15.00.0 10 0 0.0 5,530 97,450 17.6 41.1 10 0 1.0 4,401 14,107 3.2 70.5 10 501.0 2,423 5,445 2.2 74.0 10 200 1.0 1,956 4,021 2.1 73.8 10 500 1.01,705 3,367 2.0 72.3 10 1000 1.0 1,601 3,086 1.9 70.6 CGTase:Cyclomaltodextrin glucanotransferase

As is evident from the results in FIG. 19 and Table 21, Mw/Mn, the valueof dividing the weight-average molecular weight with the number averagemolecular weight of the branched α-glucan, prepared by using theα-glucosyltransferase of the present invention and 1.0 unit/g-solid ofCGTase in combination, was decreased to 3.2. In the case of adding 1,000units/g-solid of isoamylase to the reaction system (Symbol “e” in FIG.19), Mw/Mn was further decreased to 1.9. The WSDF content in thebranched α-glucan, prepared by using the α-glucosyltransferase of thepresent invention and CGTase in combination, was increased to about 70%(w/w), and the contents were kept the relatively high values by addingisoamylase to the reaction systems.

From these results, it was revealed that the branched α-glucan with asignificantly lowered molecular weight and significantly increased WSDFcontent can be produced by allowing the α-glucosyltransferase of thepresent invention, isoamylase, and CGTase to act in combination on thepartial starch hydrolyzate.

Experiment 19 Functions of the Branched α-Glucan

The branched α-glucan, prepared in Experiment 18-4 by using 10units/g-solid of the α-glucosyltransferase, 50 units/g-solid ofisoamylase, and 1.0 unit/g-solid of CGTase in combination, was selectedas the branched α-glucan with the highest WSDF content and a relativelylow molecular weight; and the digestibility, structural characteristics,and functions of the branched α-glucan was investigated.

Experiment 19-1 Purification of the Branched α-Glucan Prepared by Usingα-Glucosyltransferase, Isomalyase, and CGTase in Combination

A reaction mixture containing the branched α-glucan was prepared by thesame reaction in Experiment 18-4 using 10 units/g-solid of theα-glucosyltransferase, 50 units/g-solid of isoamylase, and 1.0unit/g-solid of CGTase in combination. After removing insolublesubstances from the reaction mixture by filtration, the resultingfiltrate was decolored and deionized using “DIAION™ SK-1B” and “DIAION™WA30”, both ion-exchange resins commercialized by Mitsubishi ChemicalCorporation, Tokyo, Japan, and “IRA 411”, an anion exchange resincommercialized by Organo Corporation, Tokyo, Japan; and then filtratedusing a membrane and concentrated using an evaporator. By the aboveprocedure, an aqueous solution, containing the branched α-glucan, with aconcentration of 30% was obtained in a yield of 85.8%, on a dry solidbasis, from the material starch. According to the method in Experiment4-2, the methylation analysis of the resulting branched α-glucan wascarried out and the result is shown in Table 22. In addition, the resultof the molecular weight distribution analysis, obtained by thegel-filtration HPLC described in Experiment 4-1, the WSDF contents,measured by the Enzyme-HPLC method described in Experiment 3, and theresult of isomaltodextranase digestion, obtained by the method describedin Experiment 4-2, of the branched α-glucan are summarized in Table 23.

TABLE 22 Composition (Peak Partially methylated product CorrespondingGlc** area %) 2,3,4,6-Tetramethylated Non-reducing end Glc 13.7 product3,4,6-Trimethylated product Glc involving 1,2-linkage 02,4,6-Trimethylated product Glc involving 1,3-linkage 1.62,3,6-Trimethylated product Glc involving 1,4-linkage 22.82,3,4-Trimethylated product Glc involving 1,6-linkage 54.72,4-Dimethylated product Glc involving 1,3,6-linkage 2.42,3-Dimethylated product Glc involving 1,4,6-linkage 4.8 *Glucoseresidue

TABLE 23 Molecular weight distribution Isomaltose Number Weight- contentaverage average after molecular molecular isomalto- weight weight WSDFdextranase (Mn) (Mw) content digestion (Dalton) (Dalton) Mw/Mn (%, w/w)(%, w/w) 2,400 5,480 2.3 68.6 36.4

As is evident from the results in Table 22 and 23, in the methylationanalysis of the branched α-glucan, the partially methylated productscomprised 2,3,6-trimethylated product and 2,3,4-trimethylated product ina ratio of 1:2.4, and the total content of 2,3,6-trimethylated productand 2,3,4-trimethylated product was 77.5%. The contents of2,4,6-trimethylated product and 2,4-dimethylated product were 1.6% and2.4%, respectively, in the partially methylated products. The branchedα-glucan showed the weight-average molecular weight of 5,480 daltons andMw/Mn of 2.3. The WSDF content of the branched α-glucan, measured by theEnzyme-HPLC method, was 68.6%. From the branched α-glucan, 36.4% (w/w)of isomaltose was formed by the isomaltodextranase digestion.

In order to evaluate the usefulness of the branched α-glucan of thepresent invention, cariogenicity, digestibility, effects on blood-sugarlevel and insulin level, and acute toxicity of the branched α-glucanwere investigated in the following Experiment 19-2 to 19-7 using thebranched α-glucan prepared in Experiment 19-1.

Experiment 19-2 Acid-Fermentation Test of the Branched α-Glucan UsingCariogenic Bacteria

According to the method of Ohshima et al. described in Infection andImmunity, vol. 39, pp. 43-49 (1983), an acid-fermentation test of thebranched α-glucan, obtained in Experiment 19-1, was carried out usingcariogenic bacteria. Two bacterial strains, Streptococcus sobrinus 6715and Streptococcus mutans OMZ-176, were used as cariogenic bacteria.Sucrose was used as a control saccharide, and tested by the same method.The results are in Table 24.

TABLE 24 pH S. sobrinus S. mutans Branched Branched α-glucan α-glucanTime Sucrose (Present Sucrose (Present (min) (Control) invention)(Control) invention) 0 6.7 6.7 6.8 6.8 5 4.3 5.9 6.0 6.1 15 4.0 6.1 4.66.2 30 3.9 6.1 4.4 6.2 60 3.9 6.1 4.3 6.1 90 4.0 6.1 4.2 6.1

As is evident from the results in Table 24, in the case of sucrose, thepHs of the culture broth inoculated with the cariogenic bacteria werelowered by the acid-formation. While, in the case of the branchedα-glucan of the present invention, the saccharide was not fermented toform acids by Streptococcus sobrinus and Streptococcus mutans, and thepHs of the culture broth were kept to about 6. The pH is higher than 5.5which is critical pH of decalcifying enamel of tooth. From the results,it was confirmed that the branched α-glucan of the present inventionshows a significantly low cariogenicity.

Experiment 19-3 Digestibility of the Branched α-Glucan

According to the method of Okada et al., described in Journal ofJapanese Society of Nutrition and Food Sciences, vol. 43, pp. 23-29(1990), the digestibility of the branched α-glucan by salivaryα-amylase, artificial gastric juice, pancreas amylase, and smallintestinal enzymes were investigated in vitro using the branchedα-glucan, obtained in Experiment 19-1. “PINEFIBER®”, a low-digestibledextrin commercialized by Matsutani Chemical Industries Co., Ltd.,Hyogo, Japan, was used as a control. The results are in Table 25.

TABLE 25 Digestion (%) Low-digestible Branched α-glucan dextrinDigestive enzyme (Present invention) (Control) Salivary amylase 0.0 0.3Artificial gastric juice 0.0 0.0 Pancreas amylase 0.2 2.4 Smallintestinal enzymes 16.4 41.1

As is evident from the results in Table 25, the branched α-glucan of thepresent invention was not digested by either of salivary amylase, andartificial gastric juice, and slightly digested by pancreas amylase. Thedigestion (%) of the branched α-glucan by small intestinal enzymes was16.4% and much lower than 41.4%, that of the low-digestible dextrin usedas a control. It was revealed that the digestibility of the branchedα-glucan of the present invention is much lower than the commerciallyavailable low-digestible dextrin.

Experiment 19-4 Effects of the Ingestion of the Branched α-Glucan onBlood-Sugar Level and Insulin Level

Effects of the branched α-glucan on the elevation of blood-sugar leveland insulin level were investigated using the branched α-glucan obtainedby the method in Experiment 19-1. Five male Wister rats (seven weeksold)/group were preliminary fasted for one day, and then an aqueoussolution containing the branched α-glucan was orally administrated tothe rats using a gastric sonde. The dose was set to 1.5 g-solid/kg-ratweight. Blood samples were withdrawn from the caudal vein of rats atjust before administration, 15 min-after, 30 min-after, 60 min-after,and 120 min-after from the administration. Each blood sample wascollected in a heparin-treated tube and then centrifuged at 2,000 rpmfor 10 min to obtain blood plasma. Blood-sugar level in the blood plasmawas measured by the glucose oxidase-peroxidase method, and insulin levelwas measured using a kit for measuring rat insulin, commercialized byMorinaga Institute of Biological Science, Inc., Kanagawa, Japan. Glucoseand “PINEFIBER®”, a low-digestible dextrin commercialized by MatsutaniChemical Industries Co, Ltd., Hyogo, Japan, were used as Control 1 and2, respectively. Blood-sugar level and insulin level of each test groupare in Tables 26 and 27, respectively.

TABLE 26 Blood-sugar level (mg/dl) Low-digestible Time Branched α-glucanGlucose dextrin (min) (Present invention) (Control 1) (Control 2) Justbefore  68 ± 17  76 ± 17  66 ± 15 ingestion 15 121 ± 18 115 ± 24 109 ±20 30 156 ± 29 210 ± 29 151 ± 18 60 150 ± 11 191 ± 8  150 ± 4  120 114 ±9  144 ± 29 112 ± 15 180  80 ± 10 105 ± 17  90 ± 10

TABLE 27 Insulin level (ng/ml) Low-digestible Time Branched α-glucanGlucose dextrin (min) (Present invention) (Control 1) (Control 2) Justbefore 0.14 ± 0.07 0.33 ± 0.06 0.24 ± 0.21 ingestion 15 0.73 ± 0.29 1.06± 0.44 0.46 ± 0.12 30 0.89 ± 0.26 1.46 ± 0.29 0.95 ± 0.40 60 0.60 ± 0.091.00 ± 0.26 0.48 ± 0.25 120 0.63 ± 0.20 0.67 ± 0.21 0.31 ± 0.22 180 0.43± 0.12 0.47 ± 0.04 0.27 ± 0.1 

As is evident from the results in Tables 26 and 27, it was revealed thatin the case of the branched α-glucan of the present invention, theelevation of blood-sugar level and insulin level was inhibited as in thecase of the commercially available low-digestible dextrin in comparisonwith the case of glucose.

Experiment 19-5 Acute Toxicity Test

By using mice, the branched α-glucan obtained by the method inExperiment 19-1 was orally administrated to the mice for its acutetoxicity test. As a result, it was revealed that the branched α-glucanof the present invention is a safe substance with a relatively lowtoxicity, and that no mouse died even when administrated with it at thehighest possible dose. The LD₅₀ value of the branched α-glucan was 5g/kg-mouse weight or higher.

Experiment 20 Inhibitory Effect of the Branched α-Glucan on theElevation of Blood-Sugar Level

In Experiment 19-4, it was revealed that the elevation of blood-sugarlevel and insulin value is inhibited by ingesting the branched α-glucanin comparison with the case of ingesting glucose. Based on the results,effect of the ingestion of the branched α-glucan on the elevation ofblood-sugar level was further investigated in detail.

Experiment 20-1 Effects of the Ingestion of the Branched α-Glucan onBlood-Sugar Level and Insulin Level when the Branched α-Glucan isIngested Together with Partial Starch Hydrolyzate

The effect of the branched α-glucan on the elevation of blood-sugarlevel after ingesting “PINEDEX® #1”, a partial starch hydrolyzatecommercialized by Matsutani chemical Industries Co., Ltd., Hyogo, Japan,was investigated. Three groups of five Wister male rats (seven-weeksold)/group, purchased from Charles river Laboratories Japan Inc.,Kanagawa, Japan, are preliminary reared for one week using “AIN-93G”, afeed for rats prepared in Hayashibara Biochemical Laboratories Inc.,Okayam, Japan, (Ref. Journal of Nutrition, vol. 123, pp. 1939-1951(1993); hereinafter, called as “purified diet”) and then fasted for oneday. Successively, an aqueous solution prepared by dissolving thepartial starch hydrolyzate and the branched α-glucan was orallyadministrated to the rats using a gastric sonde. The dose of the partialstarch hydrolyzate was set to 1.5 g-solid/kg-body weight. The doses ofthe branched α-glucan were set to 0.15, 0.30, or 0.75 g-solid/kg-bodyweight for each group. Blood samples were withdrawn from the caudal veinof rats at just before administration, 15 min-after, 30 min-after, 60min-after, 120 min-after, 180 min-after, and 240 min-after from theadministration. Each blood sample was collected in a heparin-treatedtube and then centrifuged at 2,000 rpm for 10 min to obtain bloodplasma. The blood-sugar level and the insulin level in the blood plasmawere measured by the method described in Experiment 19-4. As Control 1,five rats (one group) were administrated with 1.5 g/kg-body weight ofthe partial starch hydrolyzate. As Control 2, “FIBERSOL®-2”, alow-digestible dextrin commercialized by Matsutani Chemical IndustriesCo., Ltd., Hyogo, Japan, was used as a substitute of the branchedα-glucan and the low-digestible dextrin was administrated to two groupsof rats (five rats/group) together with the partial starch hydrolyzate.The doses of the low-digestible dextrin were set to 0.15 and 0.75g-solid/kg-body weight for the two groups of rats, respectively. Theblood-sugar level, AUC value (area under the blood concentration-timecurve) of blood-sugar level, insulin level, and AUC value of insulinlevel of each groups are in Tables 28, 29, 30, and 31, respectively.

TABLE 28 Blood-sugar level (mg/dl) Branched α-glucan Low-digestibledextrin Partial starch (Present invention) (Control 2) Time hydrolyzateonly (g/kg-body weight) (g/kg-body weight) (min) (Control 1) 0.15 0.300.75 0.15 0.75 Just before 50 ± 3  55 ± 10  60 ± 13 63 ± 4 56 ± 6 59 ± 3ingestion 15 124 ± 18 109 ± 14 105 ± 4   96 ± 4* 107 ± 14 120 ± 5  30220 ± 37 171 ± 21 151 ± 11 130 ± 5* 164 ± 2  162 ± 12 60 175 ± 25 170 ±23 162 ± 9  145 ± 6* 168 ± 24 187 ± 10 120 110 ± 9  122 ± 16 135 ± 16126 ± 4* 134 ± 18 140 ± 16 180  91 ± 18  88 ± 11  99 ± 10  97 ± 5* 100 ±13 112 ± 13 240  85 ± 17  72 ± 10 75 ± 3 69 ± 9  84 ± 12 76 ± 3*Significantly different from the case of a low-digestible dextrin (0.75g/kg-body weight) (P < 0.05 or P < 0.01)

TABLE 29 AUC value of blood-sugar level (mg/dl min) Branched α-glucanLow-digestible dextrin Partial starch (Present invention) (Control 2)Time hydrolyzate only (g/kg-body weight) (g/kg-body weight) (min)(Control 1) 0.15 0.30 0.75 0.15 0.75 Just before 0.0 0.0 0.0 0.0 0.0 0.0ingestion 15  553 ± 119  410 ± 127 336 ± 90  254 ± 14*  383 ± 129 458 ±30 30 2,377 ± 402  1,690 ± 206  1,358 ± 315  1,013 ± 54* 1,577 ± 299 1,687 ± 149  60  6,796 ± 1,028 5,165 ± 23  4,270 ± 825   3,264 ± 219*4,869 ± 638  5,157 ± 457  120 12,354 ± 1,538 10,666 ± 1,385  9,612 ±1,567 76,467 ± 494* 10,555 ± 1,711 11,457 ± 1,013 180 15,372 ± 1,76013,698 ± 1,735 13,043 ± 2,306 10,585 ± 759* 14,215 ± 2,388 15,515 ±1,558 240 17,612 ± 2,624 15,229 ± 1,788 14,679 ± 3,073  11,822 ± 1,144*16,376 ± 2,428 17,637 ± 1,716 *Significantly different from the case ofa low-digestible dextrin (0.75 g/kg-body weight) (P < 0.01)

TABLE 30 Insulin level (ng/ml) Branched α-glucan Low-digestible dextrinPartial starch (Present invention) (Control 2) Time hydrolyzate only(g/kg-body weight) (g/kg-body weight) (min) (Control 1) 0.15 0.30 0.750.15 0.75 Just before 0.67 ± 0.02 0.65 ± 0.04 0.67 ± 0.06 0.67 ± 0.100.68 ± 0.05 0.67 ± 0.06 ingestion 15 1.14 ± 0.34 0.72 ± 0.10 0.80 ± 0.010.81 ± 0.08 0.94 ± 0.10 1.08 ± 0.33 30 2.54 ± 0.21 1.23 ± 0.36 1.14 ±0.30 1.04 ± 0.04 1.65 ± 0.25 1.02 ± 0.07 60 1.06 ± 0.16 0.86 ± 0.07 0.83± 0.06  0.81 ± 0.11* 1.02 ± 0.19 0.95 ± 0.05 120 0.81 ± 0.07 0.75 ± 0.030.76 ± 0.03  0.73 ± 0.03* 0.82 ± 0.06 0.79 ± 0.60 180 0.50 ± 0.03 0.69 ±0.08 0.56 ± 0.03 0.52 ± 0.26 0.57 ± 0.05 0.54 ± 0.04 240 0.51 ± 0.080.54 ± 0.05 0.53 ± 0.02  0.54 ± 0.03* 0.50 ± 0.02 0.51 ± 0.01*Significantly different from the case of a low-digestible dextrin (0.75g/kg-body weight) (P < 0.05)

TABLE 31 AUC value of insulin level (ng/ml min) Branched α-glucanLow-digestible dextrin Partial starch (Present invention) (Control 2)Time hydrolyzate only (g/kg-body weight) (g/kg-body weight) (min)(Control 1) 0.15 0.30 0.75 0.15 0.75 Just before 0.0 0.0 0.0 0.0 0.0 0.0ingestion 15  3.5 ± 2.6 0.5 ± 0.8  0.9 ± 0.5 1.1 ± 1.1 2.0 ± 1.2 3.1 ±2.7 30 21.1 ± 5.9 5.4 ± 4.3  5.4 ± 2.2 4.9 ± 3.1 11.3 ± 2.1  8.8 ± 6.160 54.9 ± 6.1 17.1 ± 10.3 14.9 ± 6.1 12.7 ± 6.9  31.0 ± 6.6  18.2 ± 8.3 120 70.7 ± 4.6 26.2 ± 11.7 22.5 ± 7.2 19.7 ± 12.8 45.7 ± 12.4 30.2 ±12.3 180 74.9 ± 6.8 30.8 ± 12.9 25.2 ± 8.6 23.0 ± 16.8 49.9 ± 13.1 34.0± 14.7 240 — 32.4 ± 13.8 — 24.2 ± 19.2 — — —: AUC value was notcalculated because the insulin level was lowered than that of justbefore ingestion.

As is evident from the results in Tables 28 to 31, it was revealed thatthe branched α-glucan of the present invention showed a dose-dependentinhibition of elevating blood-sugar level, AUC value of blood-sugarlevel, insulin level, and AUC value of insulin level when a saccharide(partial starch hydrolyzate) was loaded in comparison with the case ofadministrating the partial starch hydrolyzate only (Control 1),similarly with the case of administrating the low-digestible dextrin(Control 2). Comparing the degree of the inhibitory effects between thebranched α-glucan and the low-digestible dextrin, the branched α-glucanof the present invention shows relatively strong effects than thelow-digestible dextrin.

Experiment 20-2 Effects of the Molecular Weight of the Branched α-Glucanon Blood-Sugar Level and Insulin Level when the Branched α-Glucan isIngested Together with the Partial Starch Hydrolyzate

In Experiment 20-1, it was revealed that the branched α-glucan of thepresent invention inhibits the elevation of blood-sugar level, AUC valueof blood-sugar level, insulin level, and AUC value of insulin level whenit was ingested together with a saccharide (partial starch hydrolyzate).Successively, effect of the molecular weight of the branched α-glucan onthe inhibitory effect on blood-sugar level and insulin level wasinvestigated. Varying the kinds of material partial starch hydrolyzateand the amounts of the α-glucosyltransferase and the amylase, variousbranched α-glucans having the weight-average molecular weights, shown inTable 32, were prepared. As in the same manner in Experiment 20-1, ninegroups of five Wister male rats (seven-weeks old)/group, purchased fromCharles river Laboratories Japan Inc., Kanagawa, Japan, are preliminaryreared for one week using the purified diet and then fasted for one day.Successively, an aqueous solution prepared by dissolving the partialstarch hydrolyzate and any one of the branched α-glucan shown in Table32 was orally administrated to 8 groups of rats using a gastric sonde.The doses of the partial starch hydrolyzate and the branched α-glucanwere respectively set to 1.5 g-solid/kg-body weight and 0.75g-solid/kg-body weight for each group. To the remaining one group ofrats, 1.5 g/kg-body weight of the partial starch hydrolyzate only wasorally administrated as a control group. Blood samples were withdrawnfrom the caudal vein of rats at just before administration and 30min-after from the administration. Each blood sample was collected in aheparin-treated tube and then centrifuged at 2,000 rpm for 10 min toobtain blood plasma. The blood-sugar level and the inslin level in theblood plasma were measured and in Table 32. The timing of collectingblood sample after the administration was set to 30 min-after from theadministration because blood-sugar level and insulin level reach peaksat the timing when the partial stach hydolyzate only is administrated.

TABLE 32 Weight-average WSDF Blood-sugar level (mg/dl) Insulin level(ng/ml) molecular content Before 30 min-later Before 30 min-later SampleNo. weight (%, w/w) ingestion after ingestion ingestion after ingestionPartial starch — — 62 ± 6 225 ± 40 0.66 ± 0.02 2.78 ± 0.03 hydrolyzateonly (Control) 1 1,168 58.1 64 ± 7 173 ± 30 0.65 ± 0.07 1.86 ± 0.05 22,670 75.5  63 ± 12 144 ± 27 0.65 ± 0.05 1.36 ± 0.06 3 4,242 80.4 56 ± 9132 ± 15 0.67 ± 0.04 1.02 ± 0.07 4 25,618 72.4 61 ± 3 139 ± 15 0.59 ±0.03 1.21 ± 0.12 5 44,151 64.2 62 ± 8 142 ± 25 0.65 ± 0.03 1.31 ± 0.09 660,000 42.3 59 ± 7 157 ± 19 0.62 ± 0.05 1.74 ± 0.06 7 100,000 36.5  61 ±10 185 ± 28 0.58 ± 0.03 2.05 ± 0.05 8 200,000 30.1 58 ± 4 198 ± 22 0.65± 0.02 2.26 ± 0.05

As is evident from the results in Table 32, the branched α-glucanshaving the weight-average molecular weights in the range of 1,168 to200,000 inhibited the elevation of blood-sugar level and insulin levelwhen the partial starch hydrolyzate was orally administrated. Comparingthe degree of inhibiting the elevation of blood-sugar level and insulinlevel among the branched α-glucans, the inhibitory effect is significantin the cases of using the branched α-glucans having the molecularweights in the range of 1,168 to 60,000 (the WSDF contents in the rangeof 58.1 to 80.4% (w/w)), and is more significant in the cases of usingthe branched α-glucans having the molecular weights in the range of2,670 to 44,151 (the WSDF contents in the range of 64.2 to 80.4% (w/w)).

Experiment 20-3 Effects of a Long-Period Ingestion of the Branchedα-Glucan on Blood-Sugar Level and Insulin Level when the Partial StarchHydrolyzate is Ingested

In Experiment 20-1, it was revealed that the branched α-glucan of thepresent invention inhibits the elevation of blood-sugar level, AUC valueof blood-sugar level, insulin level, and AUC value of insulin level whenit was ingested together with a saccharide (partial starch hydrolyzate).In this experiment, effect of a long period (8 weeks) ingestion of thebranched α-glucan on the inhibition of elevating blood-sugar level, AUCvalue of blood-sugar level, insulin level, and AUC value of insulinlevel when the partial starch hydrolyzate is ingested was investigated.Five groups of five Wister male rats (seven-weeks old)/group, purchasedfrom Charles river Laboratories Japan Inc., Kanagawa, Japan, werepreliminary reared for one week using the purified diet. Then, threegroups of rats were rared for 8 weeks using three kinds of test dietsincorporated the branched α-glucan in an amount of 1, 2, or 5% (w/w),shown in Table 33. The rats were allowed to ingest the test diet andwater freely during the test period. At the point of rearing 4-weeks and8-weeks, the rats were fasted for one day and then an aqueous solutionprepared by dissolving the partial starch hydrolyzate was orallyadministrated to the rats using a gastric sonde to give a dose of 1.5g-solid/kg-body weight. Blood samples were withdrawn from the caudalvein of rats at just before administration, 15 min-after, 30 min-after,60 min-after, 120 min-after, 180 min-after, and 240 min-after from theadministration. Each blood sample was collected in a heparin-treatedtube and then centrifuged at 2,000 rpm for 10 min to obtain bloodplasma. The blood-sugar level and the inslin level in the blood plasmawere measured by the method described in Experiment 19-4. As Control 1,one group of rats (five rats/group) was reared using the purified dietonly. As Control 2, the remaining one group of rats (five rats/group)was rared using a diet with a formula in Table 33, in which a part ofcorn starch in the purified diet was substituted with“FIBERSOL-2®”, alow-digestible dextrin commercialized by Matsutani Chemical IndustriesCo., Ltd., Hyogo, Japan. Since almost the same results were obtainedfrom samples at the point of rearing 4-weeks and 8-weeks, the results ofmeasuring blood-sugar level, AUC value of blood-sugar level, insulinlevel, and AUC value of insulin level at the point of rearing 4-weeksfor each group were are in Tables 34, 35, 36, and 37, respectively.

TABLE 33 Composition (%, w/w) Low- digestible Branched α-glucan dextrinPurified (%, w/w) (%, w/w) Ingredient diet 1 2 5 5 Corn starch 39.748638.7486 37.7486 34.7486 34.7486 α-Starch 13.2 13.2 13.2 13.2 13.2 Casein20 20 20 20 20 Sucrose 10 10 10 10 10 Soybean oil 7 7 7 7 7 Cellulose 55 5 5 5 Mineral mix 3.5 3.5 3.5 3.5 3.5 Vitamin mix 1 1 1 1 1 L-Cystine0.3 0.3 0.3 0.3 0.3 Colin bitartrate 0.25 0.25 0.25 0.25 0.25t-Butyl-hydroquinone 0.0014 0.0014 0.0014 0.0014 0.0014 Branchedα-glucan 0 1 2 5 0 Low-digestible dextrin 0 0 0 0 5

TABLE 34 Blood-sugar level (mg/dl) Diet containing 5% (w/w) Dietcontaining Diet containing Diet containing Low- 1% (w/w) 2% (w/w) 5%(w/w) digestible Purified diet Branched Branched Branched dextrin Time(min) (Control 1) α-glucan α-glucan α-glucan (Control 2) Just beforeingestion 80 ± 4 66 ± 5 53 ± 4 54 ± 4  59 ± 5 15 180 ± 8  123 ± 16 70 ±8 79 ± 8*  97 ± 19 30 245 ± 11 184 ± 16 122 ± 17 120 ± 17  120 ± 14 60218 ± 13 190 ± 15 142 ± 29 126 ± 29* 161 ± 26 120 149 ± 10 162 ± 12 108± 32 101 ± 32* 154 ± 12 180 115 ± 9  120 ± 9   88 ± 19  84 ± 19* 111 ±9  240 91 ± 8  87 ± 10  71 ± 18  65 ± 18* 95 ± 4 *Significantlydifferent from the case of the diet containing 5% (w/w) low-digestibledextrin (P < 0.05 or P < 0.01)

TABLE 35 AUC value of blood-sugar level (mg/dl min) Diet containing 5%(w/w) Diet containing Diet containing Diet containing Low- 1% (w/w) 2%(w/w) 5% (w/w) digestible Purified diet Branched Branched Brancheddextrin Time (min) (Control 1) α-glucan α-glucan α-glucan (Control 2)Just before ingestion 0.0 0.0 0.0 0.0 0.0 15 751 ± 59  427 ± 158 126 ±26 184 ± 98  272 ± 69 30 2,746 ± 179  1,746 ± 299  772 ± 85 860 ± 272* 986 ± 268 60 7,303 ± 526  5,394 ± 273  3,147 ± 425  2,925 ± 510*  3,368 ± 820  120 13,525 ± 1,032 12,012 ± 1,177  7,457 ± 1,308 6,488 ±1,002*  9,141 ± 2,311 180 16,661 ± 1,216 16,520 ± 1,476 10,153 ± 1,6708,791 ± 1,450* 13,413 ± 3,479 240 18,073 ± 1,262 18,778 ± 1,717 11,771 ±1,933 10,022 ± 1,734  15,939 ± 4,382 *Significantly different from thecase of the diet containing 5% (w/w) low-digestible dextrin (P < 0.05 orP < 0.01)

TABLE 36 Insulin level (ng/ml) Diet containing 5% (w/w) Diet containingDiet containing Diet containing Low- 1% (w/w) 2% (w/w) 5% (w/w)digestible Purified diet Branched Branched Branched dextrin Time (min)(Control 1) α-glucan α-glucan α-glucan (Control 2) Just before ingestion0.61 ± 0.14 0.51 ± 0.04 0.49 ± 0.11 0.46 ± 0.07 0.53 ± 0.05 15 1.78 ±0.40 1.16 ± 0.30 0.88 ± 0.16  0.90 ± 0.18* 1.26 ± 0.23 30 3.16 ± 0.291.99 ± 0.52 1.17 ± 0.36  1.28 ± 0.31* 1.72 ± 0.31 60 1.41 ± 0.23 1.23 ±0.30 0.85 ± 0.14  0.79 ± 0.16* 1.15 ± 0.11 120 0.95 ± 0.23 0.82 ± 0.160.63 ± 0.17 0.64 ± 0.13 0.72 ± 0.86 180 0.63 ± 0.07 0.56 ± 0.09 0.58 ±0.11 0.55 ± 0.17 0.67 ± 0.09 240 0.63 ± 0.06 0.53 ± 0.07 0.47 ± 0.080.46 ± 0.07 0.62 ± 0.10 *Significantly different from the case of thediet containing 5% (w/w) low-digestible dextrin (P < 0.05 or P < 0.01)

TABLE 37 AUC value of insulin level (ng/ml min) Diet containing 5% (w/w)Diet containing Diet containing Diet containing Low- 1% (w/w) 2% (w/w)5% (w/w) digestible Purified diet Branched Branched Branched dextrinTime (min) (Control 1) α-glucan α-glucan α-glucan (Control 2) Justbefore ingestion 0.0 0.0 0.0 0.0 0.0 15  8.8 ± 3.0 4.9 ± 2.4 2.9 ± 1.43.3 ± 1.5* 5.5 ± 2.1 30 36.7 ± 5.8 20.9 ± 8.6  11.0 ± 3.1  12.7 ± 4.8* 19.9 ± 6.1  60  87.1 ± 11.2 53.9 ± 19.1 26.5 ± 5.7  29.8 ± 11.9* 46.0 ±11.5 120 121.6 ± 14.0 84.7 ± 30.0 41.5 ± 10.0 45.0 ± 20.5* 71.0 ± 14.1180 134.0 ± 17.2 95.8 ± 35.0 48.5 ± 15.2 53.9 ± 24.8* 80.7 ± 16.1 240137.8 ± 23.5 98.7 ± 36.2 51.8 ± 16.7 57.4 ± 26.7* 87.4 ± 20.0*Significantly different from the case of the diet containing 5% (w/w)low-digestible dextrin (P < 0.05 or P < 0.01)

As is evident from the results in Tables 33 to 37, it was revealed thatthe branched α-glucan of the present invention inhibited the elevationof blood-sugar level, AUC value of blood-sugar level, insulin level, andAUC value of insulin level when a saccharide (partial starchhydrolyzate) was loaded in comparison with the case of raring rats usingthe purified diet only (Control 1), similarly with the case of raringrats using the low-digestible dextrin (Control 2). It was also revealedthat the inhibitory effect is dependent on the amount of the branchedα-glucan incorporated into the test diet, and the inhibitory effect issignificant in the case of incorporating the branched α-glucan in anamount of 2% (w/w), and more significant in the case of incorporatingthe branched α-glucan in an amount of 5% (w/w). In the case of using atest diet incorporated with 5% (w/w) of the commercially availablelow-digestible dextrin, the degree of the inhibitory effect was almostequal with the case of using a test diet incorporated with 1% (w/w) ofthe branched α-glucan. From the results, it was confirmed that thebranched α-glucan of the present invention is advantageous in the effectof inhibiting the elevation of blood-sugar level, AUC value ofblood-sugar level, insulin level, and AUC value of insulin level when asaccharide (partial starch hydrolyzate) is loaded, in comparison withthe low-digestible dextrin. Comparing the blood-sugar level and insulinlevel just before ingestion among the test groups, those values in thegroups ingested the test diet incorporated with 2 or 5% (w/w) of thebranched α-glucan is lower than those in the groups ingested the controldiet or the test diet incorporated with 5% (w/w) of the low digestibledextrin. From the results, it was revealed that the branched α-glucan ofthe present invention lowered fasting blood-sugar level and insulinlevel effectively when it was used for a long period than thecommercially available low-digestible dextrin. While, the body weightsof rats were compared between the test group and control group at thepoint of raring 4 and 8 weeks but no significant difference was observedin the average body weight between the groups. From the results, it wasconsidered that the effect of inhibiting the elevation of blood-sugarlevel and insulin level by the branched α-glucan, confirmed by thisexperiment, does not influence the health of rats.

Experiment 21 Effects of the Ingestion of the Branched α-Glucan onBlood-Sugar Level and Insulin Level of Humans

From the above experiments using rats, it was revealed that theelevations of blood-sugar level and insulin level are inhibited byingesting the branched α-glucan together with the partial starchhydrolyzate in comparison with the case of ingesting the partial starchhydrolyzate only. Successively, the effects of the ingestion of thebranched α-glucan on the blood-sugar level and insulin level of humanswere investigated as follows: By using “PINEDEX #1®”, a partial starchhydrolyzate commercialized by Matsutani Chemical Industries Co., Ltd.,Hyogo, Japan, and the branched α-glucan prepared according to the methodin Example 5 described later, the elevation of blood-sugar level andinsulin level of humans were investigated. Twelve healthy malevolunteers (age 26 to 54, average age 41±1) were used as subjects, andtheir ingestion of foods except for water was restricted during 21o'clock of the previous day to 9 o'clock of the next day (time ofstarting the test). The subjects were allowed to ingest the testpreparation, prepared by dissolving 50 g-solid of the partial starchhydrolyzate into water and filling up to 200 ml, within a time frame of2 min, and the blood samples were collected at the points of just beforeingestion, 15 min-after, 30 min-after, 45 min-after, 60 min-after 90min-after, and 120 min-after the ingestion. After the one week or moreinterval, the same subjects were allowed to ingest the test preparation,prepared by dissolving 50 g-solid of the branched α-glucan into waterand filling up to 200 ml, and the blood samples were collected in thesame manner. The blood-sugar level and insulin level of each bloodsample were measured by entrusting to Medical Center of Okayama MedicalAssociation, a private clinical laboratory, Okayama, Japan. Time coursesof blood-sugar level and insulin level, AUC value of blood-sugar fromjust after ingestion to 120 min-after ingestion (AUC^(0-2 hr) (mghr/dl)), AUC value of insulin level of the period (AUC⁰⁻² hr (μUhr/dl)), and the increase of AUC values of blood-sugar level and insulinlevel of the period (AAUC^(0-2 hr)) are summarized in Table 38.

TABLE 38 Blood-sugar level (mg/dl) Insulin level (μU/ml) Partial starchPartial starch hydrolyzate Branched hydrolyzate Branched Time (min)(Control) α-glucan (Control) α-glucan Just before 87 ± 6 87 ± 1  5.7 ±2.1  4.9 ± 1.7 ingestion  15 113 ± 12 102 ± 8  22.7 ± 13.4 17.5 ± 15.3 30 132 ± 15 112 ± 10* 36.8 ± 13.3 25.6 ± 18.5  60 137 ± 21 113 ± 19*40.9 ± 14.8 22.4 ± 9.6* 120 121 ± 20 108 ± 24  39.6 ± 17.1 18.8 ± 7.3*180  90 ± 17 89 ± 12 26.9 ± 13.5 11.9 ± 4.6* 240  79 ± 17 80 ± 3  12.7 ±7.2   6.7 ± 2.0* AUC^(0-2 hr) 217 ± 23 197 ± 20* 57.3 ± 21.2 31.7 ±11.1* ΔAUC^(0-2 hr)  47.4 ± 12.8  28.5 ± 15.2* 45.8 ± 17.5 22.0 ± 8.4**Significantly different from the case of Control (P < 0.05 or P < 0.01)

As is evident from the results in Table 38, in the case of ingesting thebranched α-glucan of the present invention, it was revealed thatblood-sugar level, AUC value of blood-sugar level, insulin level, andAUC value of insulin level were significantly low in comparison with thecase of ingesting the partial starch hydrolyzate, similarly with thecase in Experiment 20 using rats.

Experiment 22 Effect of the Branched α-Glucan on the Lowering of Lipidsin Living Bodies

From the results in Experiments 19-4 and 21, it was revealed that theelevation of blood-sugar level and insulin level was inhibited byingesting the branched α-glucan. Successively, the effect of theingestion of the branched α-glucan on the amount of lipids in livingbodies was investigated.

Experiment 22-1 Effect of the Ingestion of the Branched α-Glucan on theAbsorption of Lipids

Seven-weeks old of Wister male rats, purchased from Charles riverLaboratories Japan Inc., Kanagawa, Japan, were randomly divided intofour groups, 15 rats/group, and preliminary reared for one week usingthe purified diet shown in Table 33. The branched α-glucan prepared bythe method in Example 5 described later was used in this experiment.Then, two groups of rats were reared for 4 weeks or 8 weeks using thetest diets incorporated with the branched α-glucan in an amount of 5%(w/w), shown in Table 33. The remaining two groups of rats were rearedfor 4 weeks or 8 weeks using the purified diet as control groups. Afterrearing 4 weeks or 8 weeks, rats in the test group, reared using thetest diet incorporated with the branched α-glucan, and those in thecontrol group were killed by collecting blood from postcaval vein underether anesthesia and dissected; and then the amount of lipidsaccumulated in the internal organ, the level of serum lipids, thewet-weight of intestinal mucosa, the contents in cecum, etc. wereinvestigated. The results are in Table 39. Rats were reared withmeasuring the body-weight and the feed intake at 2 or 3 days intervaland were allowed to ingest the diet and water freely during the testperiod. Rats were fasted for one night before the dessection. The bodyweight gain, feed intake, food efficacy (body weight gain/feed intake),body weight at the point of dissection, weights of organs, weight ofintestinal mucosa, weights of lipids of internal organs, weight of thecontent of cecum, moisture content of the content of cecum, and pH ofthe content of cecum are in Table 39. The levels of serum lipids arealso in Table 39. In the sreum lipids, the levels of triglyceride, totalcholesterol, and HDL-cholesterol were determined by using “TRIGLYCERIDEE-TEST WAKO”, a kit for measuring triglyceride commercialized by WakoPure Chemical Industries Ltd., Osaka, Japan, “CHOLESTEROL E-TEST WAKO”,a kit for measuring total cholesterol commercialized by Wako PureChemical Industries Ltd., Osaka, Japan, and “HDL-CHOLESTEROL E-TESTWAKO”, a kit for measuring HDL-cholesterol commercialized by Wako PureChemical Industries Ltd., Osaka, Japan, respectively. The level ofLDL-cholesterol was calculated by subtracting the value ofHDL-cholesterol from that of total cholesterol.

TABLE 39 4 weeks-rearing 8 weeks-rearing Diet containing Diet containing5% (w/w) 5% (w/w) Purified diet Branched Purified diet BranchedMeasurement (Control) α-glucan (Control) α-glucan Food efficacy Bodyweight gain (g) 107.7 ± 9.0  109.0 ± 16.9  174.1 ± 21.2  171.8 ± 11.6 Intake (g) 486.7 ± 32.2  488.7 ± 53.1  938.2 ± 43.6  962.1 ± 80.7 Efficacy 0.22 ± 0.01 0.22 ± 0.01 0.19 ± 0.02 0.18 ± 0.02 Weight of Bodyweight (g) 348.0 ± 11.6  345.0 ± 24.9  429.2 ± 22.6  424.6 ± 13.1 internal organ Liver 9.76 ± 0.85 9.59 ± 1.49 10.44 ± 0.74  10.42 ± 0.98 Kidney 1.28 ± 0.11 1.23 ± 0.19 2.84 ± 0.30 2.43 ± 0.89 Spleen 0.86 ±0.09 0.82 ± 0.10 0.90 ± 0.08 0.85 ± 0.06 Weight of Jejunal mucosa (g)1.06 ± 0.16  1.31 ± 0.25* 1.26 ± 0.14  1.55 ± 0.23** intestinal Ilealmucosa (g) 0.96 ± 0.22  1.15 ± 0.12* 1.10 ± 0.22  1.51 ± 0.20** mucosaCecal tissue (g) 0.99 ± 0.18 1.03 ± 0.16 1.11 ± 0.20  1.46 ± 0.21**Weight of Around mesenterium (g) 4.37 ± 1.40 4.49 ± 0.84 6.02 ± 1.196.99 ± 1.37 lipids in Around kidney (g) 6.24 ± 3.36 4.15 ± 0.98 8.05 ±2.69  4.74 ± 1.70* internal organ Around testis (g) 5.70 ± 1.86  3.32 ±1.12* 9.25 ± 2.23  3.68 ± 0.84** Contents in Weight (g) 2.17 ± 0.70 2.17± 0.70 2.37 ± 0.73 2.39 ± 0.74 cecum Moisture content (%) 76.8 ± 3.0 76.3 ± 4.0  76.7 ± 3.1  77.2 ± 3.7  pH 8.75 ± 0.23 8.59 ± 0.19 8.70 ±0.35  8.28 ± 0.48* Serum lipid Triglyceride (mg/dl) 55.7 ± 11.7 56.9 ±14.9 58.2 ± 13.8 47.2 ± 10.5 Total cholesterol (mg/dl) 68.1 ± 11.3 59.2± 10.4 62.1 ± 10.2 54.7 ± 11.2 HDL-Cholesterol (mg/dl) 53.1 ± 7.4 47.4 ±9.3  37.6 ± 5.0  33.5 ± 5.5  LDL-Cholesterol (mg/dl) 15.0 ± 4.9  11.8 ±3.1  24.4 ± 6.1  21.2 ± 5.5  *Significantly different from the case ofControl (P < 0.05) **Significantly different from the case of Control (P< 0.01)

As is evident from the results in Table 39, in the case of allowing ratsto ingest the test diet incorporated with 5% (w/w) of the branchedα-glucan of the present invention, the weights of lipids around kidneyand testis were lower at the point of rearing for 4 weeks in comparisonwith the case of allowing rats to ingest the purified diet only. At thepoint of rearing for 8 weeks, the weights of lipids around kidney andtestis were, particularly, the weight of lipids around testis wassignificantly lower than those of control. Also, the weight ofintestinal mucosa was significantly increased in the test group at thepoint of rearing for 4 and 8 weeks, and more significantly at the pointof rearing for 8 weeks. Further, the pH of the content in cecum in thetest group was significantly lowered at the point of rearing for 8weeks. Regarding the level of serum lipids, the value of triglyceride inthe test group tended to decrease at the point of rearing 8 weeks, andthe values of total cholesterol and LDL-cholesterol tended to decreaseat the points of rearing 4 and 8 weeks. Other results except for thosedescribed above were almost equal between the test group and the controlgroup. The level of organic acids in cecum was not different between thetest group and the control group (data not shown). These resultsdescribed above indicate that the branched α-glucan of the presentinvention exercises the effect of lowering lipids in living bodies.Further, the weight of intestinal mucosa was increased in the testgroup. From the result, it is suggested that the thickening ofintestinal mucosa accompanying the increase of mucin secration, thedecrease of digestive enzyme activities caused by the thickening, andthe inhibition or delay of the digestion or absorption of glucose andlipids play important roles on the inhibition of excess accumulation oflipids and enhancement of carbohydrate tolerance, confirmed byExperiments 20 and 21.

Experiment 22-2 Effects of the Molecular Weight of the Branched α-Glucanon the Inhibition of Excess Accumulation of Lipids in Living Bodies

In Experiment 22-1, it was revealed that the ingestion of the branchedα-glucan of the present invention inhibits the excess accumulation oflipids in living bodies. Successively, effect of the weight-averagemolecular weight of the branched α-glucan on the inhibitory effect wasinvestigated. Eight kinds of test diets were prepared by incorporating 8kinds of branched α-glucan with different weight average molecularweight, used in Experiment 20-2, into the purified diet to give acontent of 5% (w/w). Forty-five Wister male rats (seven-weeks old),purchased from Charles river Laboratories Japan Inc., Kanagawa, Japan,were divided into 9 groups, 5 rats/group, and preliminary reared for oneweek using the purified diet. Eight groups of rats were reared for 8weeks using any one of the test diets (test diet Nos. 1 to 8)incorporated with the branched α-glucan with different weight-averagemolecular weight, shown in Table 40. The remaining one group of rats wasreared using the purified diet for 8 weeks as a control group. Afterrearing 8 weeks, 8 groups of rats as test groups and one group of ratsas control group were killed by collecting blood under ether anesthesia;and then, the weights of lipids (wet-weight) around mesenterium, kidney,and testis, and the levels of triglyceride and total cholesterol weremeasured by the same method in Experiment 22-1. The results are in Table40.

TABLE 40 Branched α-glucan incorporated into the purified dietWeight-average WSDF Weight of lipids in internal organ (g) Serum lipids(mg/dl) Test diet No. molecular content Around Around Around Total usedfor breeding weight (%, w/w) mesenterium kidney testis Triglyceridecholesterol Reared Using — — 6.22 ± 1.09 8.65 ± 2.79 8.95 ± 2.16  60.1 ±12.6 63.1 ± 9.8 purified diet only (Control) 1 1,168 58.1 6.24 ± 1.486.28 ± 1.79 7.28 ± 2.32 49.8 ± 9.1 54.7 ± 8.9 2 2,670 75.5 5.92 ± 1.295.12 ± 1.43 4.65 ± 1.01 45.4 ± 8.2 48.5 ± 9.1 3 4,242 80.4 5.64 ± 1.184.43 ± 1.55 3.26 ± 0.72  46.5 ± 11.5  50.1 ± 10.3 4 25,618 72.4 5.53 ±1.32 4.86 ± 1.75 4.32 ± 1.25 47.1 ± 7.8 49.8 ± 8.9 5 44,151 64.2 5.78 ±1.57 5.36 ± 1.88 5.89 ± 1.37 50.1 ± 6.9  53.2 ± 10.1 6 60,000 42.3 6.01± 1.41 5.86 ± 1.77 6.45 ± 1.96 52.9 ± 9.1 55.3 ± 9.4 7 100,000 36.5 6.35± 1.85 6.81 ± 2.06 7.22 ± 2.08 57.7 ± 8.6  58.7 ± 12.3 8 200,000 30.16.29 ± 1.98 7.45 ± 2.26 8.05 ± 2.13 58.3 ± 9.9 60.9 ± 8.2

As is evident from the results in Table 40, in the case of allowing ratsto ingest the branched α-glucan of the present invention, with differentweight-average molecular weight, the weights of lipids in internalorgans and in serum were lowered in any one of the test groups incomparison with those of control group. From the results, it wasrevealed that the branched α-glucan of the present invention inhibitsthe increase of the weights of lipids in internal organs and in serum.Comparing the degree of the inhibitory effect among the branchedα-glucan with different weight-average molecular weight, the inhibitoryeffect is significant in the case of using the branched α-glucans withthe molecular weights in the range of 2,670 to 44,151, and is moresignificant in the cases of using the branched α-glucans with themolecular weights in the range of 2,670 to 25,618.

From the results in Experiments 19-2 to 19-5, it was revealed that thebranched α-glucan of the present invention has low-cariogenic andlow-digestible characteristics and can be advantageously used as alow-calorie WSDF. Further, from the results in Experiments 20 to 22, itwas revealed that the branched α-glucan of the present invention can beused as agents for inhibiting the elevation of blood-sugar level and forlowering the lipids in living bodies.

The following Examples 1 and 2 explain the process for producing theα-glucosyltransferase of the present invention. Examples 3 to 6 explainthe process for producing the branched α-glucan of the presentinvention. Example 7 explains physicochemical properties of the branchedα-glucan of the present invention. Example 8 explains aquality-improving agent containing the α-glucosyltransferase of thepresent invention. Further, Examples 9 to 22 explain compositionsprepared by incorporating the branched α-glucan of the presentinvention.

Example 1

According to the method in Experiment 5, Bacillus circulans PP710 (FERNBP-10771) was cultivated using a fermenter for about 24 hours. Aftercompletion of the cultivation, the culture supernatant was collected bycentrifuging culture broth and admixed with ammonium sulfate to give a80% saturation and allowed to stand at 4° C. for 24 hours. The resultingprecipitate was collected by centrifugation and dissolved in 20 mMacetate buffer, pH 6.0. Then, the solution was dialyzed against the samebuffer and concentrated using a membrane to make into a concentratedcrude enzyme solution. The α-glucosyltransferase activity of theconcentrated crude enzyme solution was 200 units/ml. The concentratedcrude enzyme solution also contained about 25 units/ml of amylaseactivity. The crude enzyme solution can be advantageously used forproducing the branched α-glucan of the present invention from amylaceoussubstrates and used as a quality-improving agent for amylaceoussubstances in foods and beverages.

Example 2

According to the method in Experiment 8, Arthrobacter globiformis PP349(FERM BP-10770) was cultivated using a fermenter for about 24 hours.After completion of the cultivation, culture supernatant was collectedby centrifuging culture broth and admixed with ammonium sulfate to givea 80% saturation and stand at 4° C. for 24 hours. The resultingprecipitate was collected by centrifugation and dissolved in 20 mMacetate buffer, pH 6.0. Then, the solution was dialyzed against the samebuffer and concentrated using a membrane to make into a concentratedcrude enzyme solution. The α-glucosyltransferase activity of theconcentrated crude enzyme solution was 50 units/ml. The crude enzymesolution can be advantageously used for producing the branched α-glucanof the present invention from amylaceous substrates and used as aquality-improving agent for amylaceous substances in foods andbeverages.

Example 3

“PINEDEX® #100”, a partial starch hydrolyzate commercialized byMatsutani Chemical Industries Co., Ltd., Hyogo, Japan, was dissolved inwater to give a concentration of 30% (w/w) and the pH of the solutionwas adjusted to 6.0. The concentrated crude enzyme solution, obtained bythe method in Example 1, was admixed with the above solution to give anα-glucosyltransferase activity of 10 units/g-dry solid of substrate, andfollowed by the enzyme reaction at 40° C. for 48 hours. After completionof the reaction, the reaction mixture was heated at 95° C. for 10minutes, cooled, and then filtrated. According to the conventionalmethods, the resulting filtrate was decolored using activated charcoal,deionized using H- and OH-form ion-exchanger resins, and concentrated toobtain the branched α-glucan solution with a concentration of 50% (w/w).On the methylation analysis of the branched α-glucan, a ratio of2,3,6-trimethylated product and 2,3,4-trimethylated product was 1:1.3,and the total content of 2,3,6-trimethylated product and2,3,4-trimethylated product was 70.3% in the partially methylatedproducts. Further, contents of 2,4,6-trimethylated product and2,4-dimethylated product were 3.0% and 4.8%, respectively, in thepartially methylated products. The weight-average molecular weight ofthe branched α-glucan was 6,220 daltons and the value of dividing theweight-average molecular weight with the number average molecular weight(Mw/Mn) was 2.2. In addition, 35.1% (w/w) of isomaltose, on a dry solidbasis of hydrolyzate, was formed from the branched α-glucan byisomaltodextranase digestion. The WSDF content of the branched α-glucanwas 75.8% (w/w) by Enzyme-HPLC method. Since the product has anon-cariogenicity, hardly digestible property, and adequate viscosity,it can be advantageously used in various compositions such as foods andbeverages, cosmetics, and pharmaceuticals as a WSDF, substitute of fatfor foods, foods and beverages for diet, quality-improving agent,stabilizer, excipient, thickener, and filler.

Example 4

A tapioca starch was prepared into a 30% (w/v) starch suspension,admixed with calcium carbonate to give a concentration of 0.1% (w/w),adjusted to pH 6.5, and admixed with 0.2%/g-starch of “THERMAMYL™ 60 L”,an α-amylase commercialized by Novo Industries A/S, Copenhagen, Denmark,and then incubated at 95° C. for 15 min. After autoclaving at 120° C.for 10 min, the reaction mixture was cooled to about 40° C. Theliquefied starch solution was admixed with 10 units/g-dry solid starchof the concentrated crude enzyme solution containingα-glucosyltransferase, prepared by the method in Example 2, and oneunit/g-dry solid starch of CGTase from Bacillus stearothermophilus,commercialized by Hayashibara Biochemical Laboratories Inc., Okayama,Japan, and followed by the enzymatic reaction at pH 6.0 and 40° C. for72 hours. After heating the reaction mixture at 95° C. for 10 minutes,it was cooled and filtrated. According to the conventional methods, theresulting filtrate was decolored using activated charcoal, deionizedusing H- and OH-form ion-exchanger resins, concentrated, and spray-driedto obtain the powdery branched α-glucan. On the methylation analysis ofthe branched α-glucan, a ratio of 2,3,6-trimethylated product and2,3,4-trimethylated product was 1:1.6, and the total content of2,3,6-trimethylated product and 2,3,4-trimethylated product was 80.0% inthe partially methylated products. Further, contents of2,4,6-trimethylated product and 2,4-dimethylated product were 1.4% and1.7%, respectively, in the partially methylated products. Theweight-average molecular weight of the branched α-glucan was 10,330daltons and the value of dividing the weight-average molecular weightwith the number average molecular weight (Mw/Mn) was 2.9. In addition,40.7% (w/w) of isomaltose, on a dry solid basis of hydrolyzate, wasformed from the branched α-glucan by isomaltodextranase digestion. TheWSDF content of the branched α-glucan was 68.6% (w/w) by Enzyme-HPLCmethod. Since the branched α-glucan has a non-cariogenicity, hardlydigestible property, and adequate viscosity, it can be advantageouslyused in various compositions such as foods and beverages, cosmetics, andpharmaceuticals as a WSDF, substitute of fat for foods, foods andbeverages for diet, quality-improving agent, stabilizer, excipient,thickener, and filler.

Example 5

To 27.1% (w/w) of liquefied corn starch (hydrolysis: 3.6%), sodiumbisulfite and calcium chloride were added to give final concentrationsof 0.3% (w/w) and 1 mM, respectively. Then the solution was cooled to50° C. and admixed with 11.1 units/g-solid of the concentrated crudeenzyme solution, prepared by the method in Example 1, and followed bythe enzyme reaction at pH 6.0 and 50° C. for 68 hours. After heating thereaction mixture at 80° C. for 60 minutes, it was cooled and filtrated.According to the conventional methods, the resulting filtrate wasdecolored using activated charcoal, deionized using H- and OH-formion-exchanger resins, concentrated, and spray-dried to obtain thepowdery branched α-glucan. On the methylation analysis of the branchedα-glucan, a ratio of 2,3,6-trimethylated product and 2,3,4-trimethylatedproduct was 1:2.5, and the total content of 2,3,6-trimethylated productand 2,3,4-trimethylated product was 68.4% in the partially methylatedproducts. Further, contents of 2,4,6-trimethylated product and2,4-dimethylated product were 2.6% and 6.8%, respectively, in thepartially methylated products. The weight-average molecular weight ofthe branched α-glucan was 4,097 daltons and the value of dividing theweight-average molecular weight with the number average molecular weight(Mw/Mn) was 2.1. In addition, 35.6% (w/w) of isomaltose, on a dry solidbasis of hydrolyzate, was formed from the branched α-glucan byisomaltodextranase digestion. The WSDF content of the branched α-glucanwas 79.4% (w/w) by Enzyme-HPLC method. Since the branched α-glucan has anon-cariogenicity, hardly digestible property, and adequate viscosity,it can be advantageously used in various compositions such as foods andbeverages, cosmetics, and pharmaceuticals as a WSDF, substitute of fatfor foods, foods and beverages for diet, quality-improving agent,stabilizer, excipient, thickener, and filler.

Example 6

Except for using the purified α-glucosyltransferase from Bacilluscirculans PP710, FERM BP-10771, prepared by the method in Experiment 6,instead of the concentrated crude enzyme preparation, and using 1,000units/g-solid of isoamylase from Pseudomonas amyloderamosa,commercialized by Hayashibara Biochemical Laboratories Inc., Okayama,Japan; the powdery branched α-glucan was obtained according to themethod in Example 5. On the methylation analysis of the branchedα-glucan, a ratio of 2,3,6-trimethylated product and 2,3,4-trimethylatedproduct was 1:4, and the total content of 2,3,6-trimethylated productand 2,3,4-trimethylated product was 67.9% in the partially methylatedproducts. Further, contents of 2,4,6-trimethylated product and2,4-dimethylated product were 2.3% and 5.3%, respectively, in thepartially methylated products. The weight-average molecular weight ofthe branched α-glucan was 2,979 daltons and the value of dividing theweight-average molecular weight with the number average molecular weight(Mw/Mn) was 2.0. In addition, 40.6% (w/w) of isomaltose, on a dry solidbasis of hydrolyzate, was formed from the branched α-glucan byisomaltodextranase digestion. The WSDF content of the branched α-glucanwas 77% (w/w) by Enzyme-HPLC method. Since the branched α-glucan has anon-cariogenicity, hardly digestible property, and adequate viscosity,it can be advantageously used in various compositions such as foods andbeverages, cosmetics, and pharmaceuticals as a WSDF, substitute of fatfor foods, foods and beverages for diet, quality-improving agent,stabilizer, excipient, thickener, and filler.

Example 7

According to the conventional methods, physicochemical properties of thebranched α-glucan prepared in Example 5 were investigated and theresults are summarized in Table 41 as an example of properties of thebranched α-glucan of the present invention.

TABLE 41 Aspect Tasteless and odorless white amorphous powder SolubilityNot soluble in alcohol, acetone, hexane, benzene, ethyl-acetate, carbontetrachloride, chloroform, and ether. Soluble in water, formamide, anddimethyl sulfoxide pH of aqueous solution Slightly acidic Componentsugar Glucose only Specific optical rotation +194.1° to +194.4°(Concentration, 20° C.) Color reaction Positive: Anthrone-sulfatereaction, Phenol-sulfate reaction Negative: Biuret reaction,Lowry-Foline reaction, Elson-Morgan reaction Melting point Not showingclear melting point Methylation analysis Showing the presence of glucoseresidues involving non-reducing end, 1,3-linkage, 1,4-linkage,1,6-linkage, 1,3,6-linkage, and 1,4,6-linkage Infrared resonance Showinga characteristic absorption to spectrum α-anomer of D-glucose around 844cm⁻¹ C-NMR spectrum Showing a characteristic signal to α-1,6 linkagearound 68 ppm Enzymatic digestibility Forming isomaltose by dextranasetreatment

Example 8 Quality-Improving Agent

Four hundred parts by weight of “FINETOSE®”, an anhydrous maltosecommercialized by Hayashibara Shoji Inc., Okayama, Japan, 200 parts byweight of “TREHA®”, trehalose commercialized by Hayashibara Shoji Inc.,Okayama, Japan, and two parts by weight of purifiedα-glucosyltransferase solution, prepared from Bacillus circulans PP710(FERM BP-10771) by the method in Example 6, were mixed to homogeneityand dried by conventional circulation drying to make into an enzymepreparation comprising α-glucosyltransferase. The product can be usedfor modifying amylaceous substances and inhibiting the retrogradation ofstarch by incorporating into amylaceous substances for producing foodsand beverages. Therefore, it can be advantageously used as aquality-improving agent, particularly, as a starch-retrogradationinhibiting agent.

Example 9 “Mochi” (Rice Cake)

Five hundred parts by weight of “shiratamako” (rice flour) and 500 partsby weight of “joshinko” (rice flour) were mixed to homogeneity, then,700 parts by weight of water was admixed with the mixture and steamedwith vapor for 40 minutes. Successively, the steamed rice flour waskneaded into dough using “ACM20LVW”, a mixer commercialized by Aicoh,Saitama, Japan. After cooling the dough to about 55° C., 360 parts byweight of sucrose and 240 parts by weight of “TREHA®”, trehalosecommercialized by Hayashibara Shoji Inc., Okayama, Japan, were admixedwith the dough. Then, the α-glucosyltransferase of the presentinvention, purified by the method in Experiment 6, was admixed with theabove mixture to give a final enzyme activity of 50 units/g-starchysubstance by dividing to four times. Successively, the mixture wasfurther kneaded for three minutes, and then shaped by filling the doughinto a plastic container with the internal diameter of 60 mm and theheight of 22 mm, and cooled and preserved. The product is a “mochi”(rice cake) with a high quality, soft texture, and extendibility becauseamylaceous substance in the dough is converted into branched α-glucan bythe action of α-glucosyltransferase and it inhibits the retrogradationof starch.

Example 10 “Ohagi” (Rice Dumpling Covered with Bean Jam)

Three hundred-fifty parts by weight of “SUNMALT®”, maltosecommercialized by Hayashibara Shoji Inc., Okayama, Japan, and 150 partsby weight of “TREHA®”, trehalose commercialized by Hayashibara ShojiInc., Okayama, Japan, were dissolved into hot water to make into asaccharide solution with a concentration of 70% (w/w), and then kept ata temperature of 55° C. Successively, 1,000 parts by weight of glutinousrice, which had been soaked in water, was steamed by the conventionalmethod using a steamer, and then cooled to 55° C. To the steamedglutinous rice, 500 parts by weight of the above saccharide solution and25 units/g-starchy substance of the α-glucosyltransferase of the presentinvention, purified by the method in Experiment 9, were admixed andstirred to homogeneity. After keeping the mixture at 45 to 50° C. forabout one hour in a heated container, it was made into “ohagi” (ricedumpling covered with bean jam) using bean jam. The product is “ohagi”with a high quality, which keeps soft texture just after preparation andshows no syneresis when thawed after refrigeration or freezing, becausegelatinized starch is converted into branched α-glucan by the action ofα-glucosyltransferase and it inhibits the retrogradation of starch.

Example 11 Sweetened Condensed Milk

Two parts by weight of the branched α-glucan, obtained by the method inExample 3, and three parts by weight of sucrose were dissolved in 100parts by weight of material milk. The resulting mixture was sterilizedby heating with a plate heater, concentrated to give a concentration of70%, and then packed in a can under a sterile condition to make into aproduct. Since the product has a mild sweetness and good flavor, it canbe advantageously used as a sweetened condensed milk rich in WSDF forseasoning fruits, coffee, cocoa, black tea, and the like.

Example 12 Lactic Acid Bacteria Beverage

One hundred seventy-five parts by weight of skim milk, 50 parts byweight of the powdery branched α-glucan, obtained by the method inExample 4, and 50 parts by weight of “NYUKA-OLIGO®”, a lactosucrose highcontent powder commercialized by Hayashibara Shoji Inc., Okayama, Japan,were dissolved into 1,500 parts by weight of water, and then theresulting mixture was sterilized at 65° C. for 30 min. After cooling themixture to 40° C., 30 parts by weight of a lactic acid bacterium wasinoculated to the mixture as a starter according to conventional method,and cultured at 37° C. for eight hours to obtain a lactic acid bacteriabeverage. The product has a satisfactory flavor and keeps the lacticacid bacterium stably because it comprises branched α-glucan as a WSDFand oligosaccharide. Further, the product is preferably used as a lacticacid bacteria beverage having a growth-promoting activity forbifidobacteria and a function-regulating activity for intestine.

Example 13 Powdery Juice

To 33 parts by weight of a powdery orange juice, produced by aspray-drying method, 10 parts by weight of a powdery branched α-glucan,obtained by the method in Example 4, 20 parts by weight of hydrouscrystalline trehalose, 20 parts by weight of anhydrous crystallinemaltitol, 0.65 part by weight of anhydrous citric acid, 0.1 part byweight of malic acid, 0.2 part by weight of 2-O-α-glucosyl-L-ascorbicacid, 0.1 part by weight of sodium citrate, 0.5 part by weight ofpullulan, and suitable amount of powdery flavor were mixed with stirringand the resulting powdery mixture was pulverized to make into a finepowdery product. Then, the powdery product was subjected to a fluidizedbed granulator and its exhaust temperature was set to 40° C. A suitableamount of a solution comprising branched α-glucan, obtained by themethod in Example 2, was sprayed as a binder on the powdery product andgranulated for 30 min and the resulting product was weighted and packedto make into a product. The product is a powdery juice with afruit-juice content of about 30%. Since the product shows no strangetaste and smell, it has a high quality and commercial value as alow-calorie juice rich in WSDF.

Example 14 Custard Cream

One hundred parts by weight of corn starch, 30 parts by weight of thesolution comprising branched α-glucan, obtained by the method in Example3, 70 parts by weight of hydrous crystalline trehalose, 40 parts byweight of sucrose, and one part by weight of sodium chloride were mixedwell, and then 280 parts by weight of whole egg was further admixed withthe mixture. Successively, 1,000 parts by weight of boiled milk wasgradually admixed with the resulting mixture and the resulting solutionwas continuously stirred on an open flame. The heating was stopped atthe point that corn starch was completely gelatinized to give atransparency. After cooling the mixture, a suitable amount of vanillaessence was admixed with the mixture, weighted, and packed to make intoa custard cream product. The product is a high quality custard creamwith a satisfactory gloss and flavor and rich in WSDF.

Example 15 “Ann” (Sweetened Bean Jam)

According to the conventional method, 10 parts by weight of materialadzuki bean was boiled in water, and removing astringents, lixivium, andwater-soluble contaminant, and made into about 21 parts by weight ofadzuki “ann” (bean j am). Then, 14 parts by weight of sucrose, 5 partsby weight of a solution comprising branched α-glucan, obtained by themethod in Example 3, and four parts by weight of water were admixed withthe above bean jam and boiled. After adding a small amount of salad oil,the bean jam was kneaded without crushing bean to make into about 35parts by weight of product. Since the product is stable bean jam withoutcolor-deterioration and syneresis and rich in branched α-glucan as aWSDF, it can be preferably used as confectionery material such as bunfilled with bean jam, bean jam cake, ice milk and the like.

Example 16 Bread

One hundred parts by weight of wheat flour, two parts by weight ofyeast, five parts by weight of sucrose, 1 part by weight of the branchedα-glucan obtained by the method in Example 4, 0.1 parts by weight ofinorganic salts and water were mixed and kneaded by the conventionalmethod. Then, the resulting dough was fermented at 26° C. for two hours,further fermented for 30 minutes, and baked.

The product show satisfactory color, a fluffy bulge, and rich in thebranched α-glucan as a WSDF. The product is bread with a high qualityshowing satisfactory elasticity and mild sweetness.

Example 17 Powdery Peptide Product

To one part by weight of “HI-NUTE S®”, 40% soybean peptides solution forfoods, commercialized by Fuji Oil Co., Ltd., Osaka, Japan, two parts byweight of the powdery branched α-glucan, obtained by the method inExample 4, was mixed and the resulting mixture was put into a plastictray, dried at 50° C. under a reduced pressure, and pulverized to makeinto a powdery peptide product. The product has a satisfactory flavorand is useful as a material for premix, low-calorie confectionaries forice dessert. Further, the product is useful as a dietary fiber andantiflaturent for a fluid diet for oral- or tube-intake.

Example 18 Cosmetic Cream

According to conventional method, two parts by weight ofpolyoxyethylenglycol mono-stearate, five parts by weight ofself-emulsified glycerin mono-stearate, two parts by weight of thepowdery branched α-glucan, obtained by the method in Example 4, one partby weight of “αG-RUTIN”, α-glucosyl rutin, commercialized by HayashibaraInc., Okayama, Japan, one part by weight of liquid paraffin, 10 parts byweight of glycerin-trioctanoate and a suitable amount of preservativewere mixed and dissolved by heating. The resulting mixture was furtheradmixed with two parts by weight of L-lactic acid, five parts by weightof 1,3-butylen glycol, and 66 parts by weight of purified water, and theresulting mixture was emulsified using a homogenizer. The homogenizedmixture was further admixed with a suitable amount of flavor and stirredto make into a cosmetic cream. The product has a satisfactorymoisture-retaining property because it comprises the branched α-glucan.The product has a satisfactory stability and can be advantageously usedas a sunburn preventive, skin-care agent and whitening agent for skin.

Example 19 Toothpaste

Forty-five parts by weight of calcium monohydrogen phosphate, 1.5 partsby weight of sodium lauryl sulfate, 25 parts by weight of glycerin, 0.5part by weight of polyoxyethylene sorbitan laurate, 15 parts by weightof the solution comprising branched α-glucan, obtained by the method inExample 3, 0.02 part by weight of saccharin, and 18 parts by weight ofwater were mixed to make into a toothpaste. The product is toothpastewhich shows a satisfactory availability without losing the washingproperty of surfactant.

Example 20 Solid Agent for a Fluid Diet

One hundred parts by weight of the powdery branched α-glucan, obtainedby the method in Example 4, 200 parts by weight of hydrous crystallinetrehalose, 200 parts by weight of a maltotetraose high content powder,270 parts by weight of powdery egg yolk, 209 parts by weight of skimmilk, 4.4 parts by weight of sodium chloride, 1.8 parts by weight ofpotassium chloride, four parts by weight of magnesium sulfate, 0.01 partby weight of thiamine, 0.1 part by weight of sodium L-ascorbate, 0.6parts by weight of vitamin E acetate, and 0.04 part by weight ofnicotinic acid-amide were mixed to make into a composition. Twenty-fivegrams each of the composition was packed into a dampproof laminatepouch, and the pouch was heat-sealed to make into a product. The productcontains WSDF, and can be advantageously used for supplying energy toliving bodies as a fluid diet to regulate the function of intestine bytaking orally or through tube into nasal cavity, stomach, and intestine.

Example 21 Tablet

To 50 parts by weight of aspirin, 14 parts by weight of powdery hydrouscrystalline trehalose and four parts by weight of the powdery branchedα-glucan, obtained by the method in Example 4, were admixed tohomogeneity. According to the conventional method, the resulting mixturewas made into tablet with 680 mg/tablet and thickness of 5.25 mm using atableting machine. The product was made by using the hardly digestiveglucan and trehalose as excipients. The product shows no hygroscopicityand satisfactory physical strength but easily disrupted in water.Further, since the branched α-glucan acts as WSDF, the tablet can beused for regulating the functions of the intestine.

Example 22 Ointment for Curing Wound

To 400 parts by weight of maltose, 50 parts by weight of a methanolsolution containing three parts by weight of iodine and 200 parts byweight of 10% (w/v) aqueous solution containing the powdery branchedα-glucan, obtained by the method in Example 4, were mixed to make intoan ointment for curing wound with an adequate extendibility and adhesiveproperty. The product shows an adequate viscosity and moisture-retainingproperty, and is an ointment with a high marketability and less changeover time. Since iodine in the product has an antimicrobial activity andmaltose in the product acts as an energy-supplement for cells, thecuring period is shortened and wound surface is cured completely.

INDUSTRIAL APPLICABILITY

Since the branched α-glucan of the present invention shows a high safetyand almost equal digestibility with a commercially availablelow-digestible dextrin, it can be advantageously used as WSDF. Further,since the branched α-glucan of the present invention exhibits effects ofinhibiting the increase of blood-sugar level and lowering lipids inliving bodies, it is useful as a health food. According to the presentinvention, the branched α-glucan, having almost equal digestibility withthe low-digestible dextrin which has been produced from starch bychemical reaction or complicated and inefficient method, can be producedefficiently in a large scale by the enzymatic reaction. The presentinvention, providing the low-digestible branched α-glucan and theprocess for producing it, is a significantly important invention thatgreatly contributes to various fields such as food and beverages,cosmetics, and pharmaceuticals.

1. A process for producing a saccharide composition comprising branched α-glucan, comprising the steps of: allowing an α-glucosyltransferase and cyclomaltodextrin glucanotransferase to act on maltose and/or α-1,4 glucan having a glucose polymerization degree of 3 or higher; and collecting the formed saccharide composition comprising branched α-glucan; said saccharide composition is characterized in that the content of water-soluble dietary fiber of the saccharide composition, obtained by applying a high performance liquid chromatography method (Enzyme-HPLC method), is 40% (w/w) or higher.
 2. The process of claim 1, wherein said saccharide composition has the following characteristics upon methylation analysis: (1) Ratio of 2,3,6-trimethyl-1,4,5-triacetyl-glucitol to 2,3,4-trimethyl-1,5,6-triacetyl-glucitol, which indicates the ratio of glucose residues with α-1,4 linkages to glucose residues with α-1,6 linkages, is in the range of 1:0.6 to 1:4; and (2) Total content of 2,3,6-trimethyl-1,4,5-triacetyl-glucitol and 2,3,4-trimethyl-1,5,6-triacetyl-glucitol, which indicates that the total content of glucose residues with α-1,4 linkages and glucose residues with α-1,6 linkages, is 60% or higher in the partially methylated glucitol acetates.
 3. The process of claim 2, wherein said saccharide composition has the following characteristics upon methylation analysis: (3) Content of 2,4,6-trimethyl-1,3,5-triacetyl-glucitol, which indicates that the content of glucose residues with α-1,3 linkage, is 0.5% or higher but less than 10% in the partially methylated glucitol acetates; and (4) Content of 2,4-dimethyl-1,3,5,6-tetraacetyl-glucitol, which indicates that the content of glucose residues with both α-1,3 and α-1,6 linkages, is 0.5% or higher in the partially methylated glucitol acetates.
 4. The process of claim 1, wherein said saccharide composition is characterized in that it forms isomaltose in an amount of 25% (w/w) or higher but 50% (w/w) or lower, on a dry solid basis, when the saccharide composition is digested by isomaltodextranase (EC 3.2.1.94).
 5. The process of claim 1, wherein said α-glucosyltransferase, which has the following physicochemical properties: (1) Molecular weight 90,000±10,000 daltons on SDS-polyacrylamide gel electrophoresis; (2) Optimum temperature 50 to 55° C. when reacted at pH 6.0 for 30 min; (3) Optimum pH pH 5.0 to 6.3 when reacted at 40° C. for 30 min; (4) Thermal stability Stable up to the temperature of 40° C. when incubated at pH 6.0 for 60 min; and (5) pH Stability Stable at least in the range of pH 4.0 to 8.0 when incubated at 4° C. for 24 hours. 